U.S. patent application number 13/387981 was filed with the patent office on 2012-06-21 for prodrugs containing an aromatic amine connected by an amido bond to a linker.
This patent application is currently assigned to ASCENDIS PHARMA A/S. Invention is credited to Julia Baron, Ulrich Hersel, Mathias Krusch, Harald Rau.
Application Number | 20120156260 13/387981 |
Document ID | / |
Family ID | 41077706 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120156260 |
Kind Code |
A1 |
Rau; Harald ; et
al. |
June 21, 2012 |
Prodrugs Containing an Aromatic Amine Connected By an Amido Bond to
a Linker
Abstract
The present invention relates to a prodrug or a pharmaceutically
acceptable salt thereof comprising a drug linker conjugate D-L,
wherein an aromatic amine containing biologically active moiety is
connected (bound) by an amido bound to a linker. The invention also
relates to pharmaceutical compositions comprising said prodrugs and
their use as medicaments.
Inventors: |
Rau; Harald; (Dossenheim,
DE) ; Baron; Julia; (Heidelberg, DE) ; Hersel;
Ulrich; (Heidelberg, DE) ; Krusch; Mathias;
(Hirschhorn, DE) |
Assignee: |
ASCENDIS PHARMA A/S
Hellerup
DK
|
Family ID: |
41077706 |
Appl. No.: |
13/387981 |
Filed: |
July 30, 2010 |
PCT Filed: |
July 30, 2010 |
PCT NO: |
PCT/EP2010/061163 |
371 Date: |
March 6, 2012 |
Current U.S.
Class: |
424/400 ;
514/367; 548/163 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/16 20180101; A61K 47/60 20170801; A61K 47/645 20170801;
A61K 47/65 20170801; A61K 47/61 20170801; A61P 25/24 20180101; A61P
25/18 20180101 |
Class at
Publication: |
424/400 ;
548/163; 514/367 |
International
Class: |
A61K 31/428 20060101
A61K031/428; A61K 9/14 20060101 A61K009/14; A61P 25/24 20060101
A61P025/24; A61P 25/00 20060101 A61P025/00; A61P 25/18 20060101
A61P025/18; C07D 277/60 20060101 C07D277/60; A61P 25/16 20060101
A61P025/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 31, 2009 |
EP |
09167029.9 |
Claims
1-19. (canceled)
20. A prodrug or a pharmaceutically acceptable salt thereof
comprising a drug linker conjugate D-L, wherein D is an aromatic
amine containing biologically active moiety; and L is a
non-biologically active linker containing i) a moiety L.sup.1
represented by formula (I), ##STR00038## wherein the dashed line
indicates the attachment of L.sup.1 to an aromatic amino group of D
by forming an amide bond; X.sup.1 is C(R.sup.1R.sup.1a) or a cyclic
fragment selected from C.sub.3-7 cycloalkyl, 4 to 7 membered
heterocyclyl, phenyl, naphthyl, indenyl indanyl, tetralinyl, or 9
to 11 membered heterobicyclyl, wherein in case X.sup.1 is a cyclic
fragment, said cyclic fragment is incorporated into L.sup.1 via two
adjacent ring atoms and the ring atom of X.sup.1, which is adjacent
to the carbon atom of the amide bond, is also a carbon atom;
X.sup.2 is a chemical bond or selected from C(R.sup.3R.sup.3a),
N(R.sup.3), O, C(R.sup.3R.sup.3a)--C(R.sup.4R.sup.4a),
C(R.sup.3R.sup.3a)--N(R.sup.4),
N(R.sup.3)--C(R.sup.4R.sup.4a)--C(R.sup.3R.sup.3a)--O, or
O--C(R.sup.3R.sup.3a), wherein in case X.sup.1 is a cyclic
fragment, X.sup.2 is a chemical bond, C(R.sup.3R.sup.3a),
N(R.sup.3) or O; optionally, in case X.sup.1 is a cyclic fragment
and X.sup.2 is C(R.sup.3R.sup.3a), the order of the X.sup.1
fragment and the X.sup.2 fragment within L.sup.1 may be changed and
the cyclic fragment is incorporated into L.sup.1 via two adjacent
ring atoms; R.sup.1, R.sup.3 and R.sup.4 are independently selected
from the group consisting of H, C.sub.1-4 alkyl and
--N(R.sup.5R.sup.5a); R.sup.1a, R.sup.2, R.sup.3a, R.sup.4a,
R.sup.5a are independently selected from the group consisting of H,
and C.sub.1-4 alkyl; R.sup.5 is C(O)R.sup.6; R.sup.6 is C.sub.1-4
alkyl; optionally, one of the pairs R.sup.1a/R.sup.4a,
R.sup.3a/R.sup.4a or R.sup.1a/R.sup.3a form a chemical bond; and
ii) a moiety L.sup.2, which is a chemical bond or a spacer, and
L.sup.2 is bound to a polymeric carrier group Z, wherein L.sup.1 is
substituted with one to four L.sup.2 moieties, provided that the
hydrogen marked with the asterisk in formula (I) is not replaced by
L.sup.2; optionally, L is further substituted.
21. The prodrug according to claim 20, wherein, in the moiety
L.sup.1 represented by formula (I), X.sup.1 is C(R.sup.1R.sup.1a),
cyclohexyl, phenyl, pyridinyl, norbonenyl, furanyl, pyrrolyl or
thienyl, wherein in case X.sup.1 is a cyclic fragment, said cyclic
fragment is incorporated into L.sup.1 via two adjacent ring atoms;
X.sup.2 is a chemical bond or selected from C(R.sup.3R.sup.3a),
N(R.sup.3), O, C(R.sup.3R.sup.3a)--O or
C(R.sup.3R.sup.3a)--C(R.sup.4R.sup.4a); R.sup.1, R.sup.3 and
R.sup.4 are independently selected from H, C.sub.1-4 alkyl or
--N(R.sup.5R.sup.5a); R.sup.1a, R.sup.3a, R.sup.4a and R.sup.5a are
independently selected from H or C.sub.1-4 alkyl; R.sup.2 is
C.sub.1-4 alkyl; R.sup.5 is C(O)R.sup.6; R.sup.6 is C.sub.1-4
alkyl;
22. The prodrug according to claim 20, wherein the moiety L.sup.1
is selected from ##STR00039## ##STR00040## ##STR00041##
##STR00042## wherein R.sup.5 is C(O)R.sup.6; R.sup.1, R.sup.1a,
R.sup.2, R.sup.3 and R.sup.6 are independently from each other
C.sub.1-4 alkyl; and L.sup.1 is substituted with one L.sup.2
moiety, preferably R.sup.2 is substituted with one L.sup.2
moiety.
23. The prodrug according to claim 20, wherein the spacer is a
fragment selected from C.sub.1-50 alkyl, C.sub.2-50 alkenyl or
C.sub.2-50 alkinyl, which fragment is optionally interrupted by one
or more groups selected from --NH--, --N(C.sub.1-4 alkyl)-, --O--,
--S--, --C(O)--, --C(O)NH--, --C(O)N(C.sub.1-4 alkyl)-,
--O--C(O)--, --S(O)--, --S(O).sub.2--, 4 to 7 membered
heterocyclyl, phenyl or naphthyl, provided that the spacer docs not
contain a nitrogen atom being in .beta.- or .gamma.-position to the
amino group containing they hydrogen marked with the asterisk in
formula (I), in case the spacer is hound to R.sup.2;
24. The prodrug according to claim 20, wherein L.sup.2 is a
chemical bond.
25. The prodrug according to claim 20, wherein the carrier group Z
is a polymer with a molecular weight .gtoreq.500 g; mol.
26. The prodrug according to claim 20, wherein L.sup.1 is
substituted with one L.sup.2 and the carrier group Z is a
biodegradable polyethylene glycol based water-insoluble
hydrogel.
27. The prodrug according to claim 20, wherein the aromatic amine
containing biologically active moiety D is derived from the
corresponding biologically active drug D-H, which is selected from
the group consisting of Abacavir, Acadesine, Acediasulfone,
Aciclovir, Actimid, Actinomycin, Adefovir, Aditeren, Afloqualone,
Aztreonam, Adefovir Dipivoxil, Adenine, Adenosine, Adenosine
monophosphate, Adenosine triphosphate, Alfuzosin, Alpiropride,
Ambasilide, Ambucaine, Ameltolide, Amethopterin, Amicycline,
Amidapsone, Amiloride, Aminoacridine, Aminoantipyrine,
Aminobenzoate, 6-Aminoflavone, 17-Aminogeldanamycin,
Aminogenistein, Aminoglutethimide, Aminohippurate,
3'-Amino-4'-methoxyflavone, Aminonimetazepam, Aminopotentidine,
Amphenidone, N-(p-Aminophenethyl)spiroperidol,
2-Amino-6(5H)-phenanthridnone, Amiphenosine, Aminophenylalanine,
Aminopterin, Aminopurvalanol A, Amfenac, Amiphenazole, Amphotalide,
Aminoisatin, Aminosalicylic Acid, Amifampridine, Amisulpride,
Amlexanox, Amonafide, Amprenavir, Amrinone, Amthamine, Anileridine,
Apraclonidine, Ascensil, Atolide, Azabon, Azacitidine, Azepexole,
Aztreonam, Basedol, Benzocaine, Batanopride, Betoxycaine,
Bleomycin, Bromfenac, Bromobuterol, Bromopride, Carbutamide,
Carumonam, Candicidin, Cefepime, Cefcapene pivoxil, Cefdaloxime,
Cefdinir, Cefditoren, Cefempidone, Cefetamet, Cefepime, Cefetecol,
Cefixime, Cefmatilen, Cefmenoxime, Cefodizime, Cefoselis,
Cefotaxime, Cefotiam, Ceftiolene, Ceftioxide, Cefpodoxime,
Cefquinome, Cefrom, Ceftazidime, Cefteram, Ceftibuten, Ceftiofur,
Ceftizoxime, Ceftriaxone, Cefuzonam, Cisapride, Clenproperol,
Chloroprocaine, Cidofovir, Cisapride, Cladribine, Clafanone,
Claforan, Clebopride, Clenbuterol, Clofarabine, Clorsulon,
Cycloclenbuterol, Cytarabine, Cytidoline, Dactinomycin,
Daniquidone, Dactinomycin, Dapsone, Daptomycin, Daraprim,
Darunavir, Dazopride, Decitabine, Declopramide, Diaminoacridine,
Dichlorophenarsine, Dimethocaine, 10-Demethoxystreptonigrin,
2,7-Dimethylproflavine, Dinalin, Dobupride, Doxazosin, Draflazine,
Emtricitabine, Entecavir, Ethacridine, Etanterol, Etoxazene,
Famciclovir, Fepratset, (.+-.)-FLA 668, Flucytosine, Fludarabine,
Folic Acid, Fosamprenavir, Ganciclovir, Gemcitabine, Gloximonam,
GSK 3B Inhibitor XII, Glybuthiazol, Hydroxymethylclenbuterol,
Hydroxyprocaine, Imiquimod, Indanocine, Iomeglamic acid, Iramine,
Isobutamben, Isoritmon, Ketoclenbuterol, Lamivudine, Lamotrigine,
Lavendamycin, Lenalidomide, Leucinocaine, Leucovorin, Lintopride,
Lisadimate, Mabuterol, Medeyol, Mesalazine, Metabutethamine,
Metabutoxycaine, Metahexamide, Methyl anthranilate, Methotrexate,
Metoclopramide, Minoxidil, Mirabegron, Mitomycin, Mocetinostat,
Monocain, Mosapride, NADH, Mutamycin, Naepaine, Naminterol,
Nelarabine, Nepafenac, Nerisopam, Nitrine, Nomifensine,
Norcisapride, Olamufloxacin, Orthocaine, Oxybuprocaine, Oximonam,
Pancopride, Parsalmide, Pathocidine, Pasdrazide, Pemetrexed,
Penciclovir, Phenazone, Phenazopyridine, Phenyl-PAS-Tebamin,
Picumeterol, Pirazmonam, Porfiromycin, Pramipexole, Prazosin,
Piridocaine, Procainamide, Procaine, Proflavine,
N-Propionylprocainamide, Proparacaine, Propoxycaine, Prucalopride,
Pyrimethamine, Questiomycin, Renoquid, Renzapride, Retigabine,
Riluzole, Rufocromomycin, S-Adenosylmethionine, Silver
sulfadiazine, Sparfloxacin, Stearylsulfamide, Streptonigrin,
Succisulfone, Sulamserod, Sulfabromomethazine, Sulfacetamide,
Sulfaclozine, Sulfaclorazole, Sulfachlorpyridazine,
Sulfachrysoidine, Sulfaclomide, Sulfacytine, Sulfadiasulfone,
Sulfadimethoxine, Sulfadimidine, Sulfadicramide, Sulfadiazine,
Sulfadoxine, Sulfaguanidine, Sulfaguanole, Sulfalene,
Sulfamerazine, Sulfamethazine, Sulfanilamidoimidazole,
Sulfanilylglycine, N-Sulfanilylnorfloxacin, Sulfathiadiazole,
Sulfamethizole, Sulfamethoxazole, Sulfametopyrazine, Sulfapyrazole,
Sulfamethoxydiazine, Sulfasymazine, Sulfatrozole, Sulfatroxazole,
Sulfamethoxypyridazine, Sulfametomidine, Sulfametrole,
Sufamonomethoxine, Sulfanilamide, Sulfaperin, Sulfaphenazole,
Sulfaproxyline, Sulfapyridine, Sulfisomidine, Sulfasomizole,
Sulfisoxazole, Suprax, Tacedinaline, Tacrine, Talampanel,
Talipexole, Tenofovir, Terazosin, Tetrahydrobiopterin,
Tetrahydrofolic acid, Thiamine, Thiazosulfone, Thioguanine,
Tigemonam, Timirdine, Trimethoprim, Triamterene, Trimethoprim,
Trimetrexate, Tritoqualine, Valaciclovir, Valganciclovir,
Veradoline, Vidarabine, Zalcitabine, and Zoxazolamine.
28. The prodrug according to claim 27, wherein D-H is
pramipexole.
29. A pharmaceutical composition comprising an effective dose of at
least one prodrug or a pharmaceutically acceptable salt thereof
according to claim 20 and a pharmaceutically acceptable
excipient.
30. The composition according to claim 29, wherein the polymer is
polyethylene glycol or a polyethylene glycol-based hydrogel,
preferably polyethylene glycol-based hydrogel microparticles with a
particle diameter of 10 to 1000 microns, preferably 15 to 100
microns.
31. The composition according to claim 29, wherein the polymer is a
polyethylene glycol-based hydrogel with a particle diameter of 10
to 1000 microns, preferably 15 to 100 microns.
32. The composition of claim 29, wherein the prodrug can be
administered by injection through a needle smaller than 0.6 mm
inner diameter.
33. The composition of claim 32, wherein the needle is smaller than
0.3 mm inner diameter.
34. The composition of claim 32, wherein the needle is smaller than
0.25 mm.
35. A method for prophylaxis and/or treatment of dopamine receptor
related diseases, including Parkinson's disease, neurological
disorders, amyotrophic lateral sclerosis, compulsive behavior,
bipolar disorders, Tourette's syndrome, depressive disorders,
treatment resistant depression, fibromyalia or restless leg
syndrome (RLS), wherein a prodrug of claim 20 is used.
36. The method according to claim 35 for prophylaxis and/or
treatment of Parkinsons's disease or RLS.
37. A method for prophylaxis and/or treatment of dopamine receptor
related diseases, including Parkinson's disease, neurological
disorders, amyotrophic lateral sclerosis, compulsive behavior,
bipolar disorders, Tourette's syndrome, depressive disorders,
treatment resistant depression, fibromyalia or restless leg
syndrome (RLS), wherein a pharmaceutical composition of claim 29 is
used.
38. The method of claim 37 for prophylaxis and/or treatment of
Parkinsons's disease or RLS.
39. A method for the synthesis of a prodrug or a pharmaceutically
acceptable salt thereof according to claim 20 comprising the step
of reacting a prodrug precursor L-Y or L.sup.1-Y with a
biologically active drug D-H to obtain the drug linker conjugate
D-L or a drug linker intermediate D-L.sup.1 by forming an amide
bond, wherein Y is a leaving group.
Description
[0001] The present invention relates to a prodrug or a
pharmaceutically acceptable salt thereof, comprising a drug linker
conjugate D-L, wherein an aromatic amine containing biologically
active moiety is connected (bound) by an amido bond to a linker.
The invention also relates to pharmaceutical compositions
comprising said prodrugs and their use as medicaments.
[0002] To enhance physicochemical or pharmacokinetic properties of
a drug in vivo, such drug can be conjugated with a carrier. If the
drug is chemically bound to a carrier and/or a linker, such systems
are commonly assigned as prodrugs. According to the definitions
provided by IUPAC (as given under
http://www.chem.qmul.ac.uk/iupac.medchem, accessed on Jul. 22,
2009), a carrier-linked prodrug is a prodrug that contains a
temporary linkage of a given active substance with a transient
carrier group that produces improved physicochemical or
pharmacokinetic properties and that can be easily removed in vivo,
usually by a hydrolytic cleavage.
[0003] The linkers employed in such carrier-linked prodrugs may be
transient, meaning that they are non-enzymatically hydrolytically
degradable (cleavable) under physiological conditions (aqueous
buffer at pH 7.4, 37.degree. C.) with half-lives ranging from, for
example, one hour to three months. On the other hand, stable
linkages such as employed in connecting moieties and spacer, are
typically non-cleavable permanent bonds, meaning that the
respective spacer or connecting moiety have a half-life of at least
six months under physiological conditions (aqueous buffer at pH
7.4, 37.degree. C.).
[0004] Suitable carriers are polymers or C.sub.8-18 alkyl groups
and can either be directly conjugated to the linker or via a
non-cleavable spacer.
[0005] The term polymer describes a molecule comprised of repeating
structural units connected by chemical bonds in a linear, circular,
branched, crosslinked or dendrimeric way or a combination thereof,
which can be of synthetic or biological origin or a combination of
both.
[0006] In addition to carrier-linked prodrugs, drugs can also be
bound to carriers in a non-covalent way, using physicochemical
formulations of drug-solvent-carrier mixtures. However, the
non-covalent approach requires a highly efficient drug
encapsulation to prevent uncontrolled, burst-type release of the
drug. Restraining the diffusion of an unbound, water soluble drug
molecule requires strong van der Waals contacts, frequently
mediated through hydrophobic moieties. Many conformationally
sensitive drugs, such as proteins or peptides, are rendered
dysfunctional during the encapsulation process and/or during
subsequent storage of the encapsulated drug. In addition, such
amino-containing drugs readily undergo side reactions with carrier
degradation products. Furthermore, dependence of the release
mechanism of the drug upon biodegradation may cause interpatient
variability.
[0007] Alternatively, the drugs may be conjugated to a carrier via
a transient linker molecule (carrier-linked prodrugs). This
approach is applied to various classes of molecules, from so-called
small molecules, through natural products up to larger proteins.
This approach is applied to various classes of molecules, from
so-called small molecules, through natural products up to larger
proteins.
[0008] Prodrug activation may occur by enzymatic or non-enzymatic
cleavage of the bond between the carrier and the drug molecule, or
a sequential combination of both, i.e. an enzymatic step followed
by a non-enzymatic rearrangement.
[0009] Enzymatically induced prodrug activation is characterized in
that the cleavage in enzyme-free in-vitro environment such as an
aqueous buffer solution, of, e.g., an ester or amide may occur, but
the corresponding rate of hydrolysis may be much too slow and not
therapeutically useful. In an in-vivo environment, esterases or
amidases are typically present and the esterases and amidases may
cause significant catalytic acceleration of the kinetics of
hydrolysis from twofold up to several orders of magnitude.
Therefore, the cleavage is predominantly controlled by the
enzymatic reaction.
[0010] A major drawback of predominantly enzymatic cleavage is
interpatient variability. Enzyme levels may differ significantly
between individuals resulting in biological variation of prodrug
activation by the enzymatic cleavage. The enzyme levels may also
vary depending on the site of administration. For instance it is
known that in the case of subcutaneous injection, certain areas of
the body yield more predictable therapeutic effects than others. To
reduce this unpredictable effect, non-enzymatic cleavage or
intramolecular catalysis is of particular interest.
[0011] Therefore, enzyme-independent autocatalytic cleavage of
carrier and biologically active moiety is preferred. In most cases
this is achieved by an appropriately designed linker moiety between
the carrier and the biologically active moiety, which is directly
attached to the functional group of a biologically active moiety
via covalent bond.
[0012] Numerous bioactive substances (drugs) contain an aromatic
amine moiety. For instance, aniline derivatives are characterized
by an amine connected to an aromatic ring. It has been subject of
research to generate prodrugs of aniline derivatives to improve on
therapeutic properties of said drug (parent compound). By
consequence, aromatic amides, such as anilides, i.e. compoundes
whose aromatic amino group is converted into an amide in order to
form a prodrug, are of interest. However, the type of linker of a
prodrug strongly influences the release rate of the aromatic amine
by cleavage of the aromatic amide fragment of such a prodrug.
[0013] Specific linker types are known in the art. For example, in
WO-A 2004/108070 there is, among others, a prodrug system based on
a N,N-bis-(2-hydroxyethyl)glycine amide (bicine) linker described.
In this system two PEG carrier molecules are linked to a bicine
molecule coupled to, for example, an amino group of the drug
molecule. By consequence, the linker only contains one amide bond,
which may be aromatic depending on the respective drug molecule
employed. In addition, the linker does not contain a secondary
amido fragment, but a tertiary amino function instead due to the
employment of a N,N-substituted spacer component. The first step in
prodrug activation is the enzymatic cleavage of the first linkages
connecting both PEG carrier molecules with the hydroxy groups of
the bicine activating group. Different linkages between PEG and
bicine are described resulting in different prodrug activation
kinetics. The second step in prodrug activation is the cleavage of
the second linkage connecting the bicine activating group to the
amino group of the drug molecule. Consequently the release of a
bicine-modified prodrug intermediate may show different
pharmacokinetic, immunogenic, toxicological and pharmacodynamic
properties as compared to the parent native drug molecule. Another
comparable bicine-based system is described in WO-A
2006/136586.
[0014] The international application PCT/EP2009/051079 also
discloses prodrugs containing a different linker type. Said prodrug
comprises a drug linker conjugate, wherein the linker is connected
to a carrier group. The linker contains a non-biologically active
linker moiety L.sup.1, which is chemically bound to the nitrogen of
the biologically active moiety by forming an amide bond. The linker
moiety L.sup.1 mandatorily contains 2 amino functions, which are
connected by an alkyl fragment containing 2 or 3 carbon atoms.
[0015] Another linker system is described in US 2005/0054612, which
relates to drug formulations that increase regional delivery of the
drugs to the cells. The respective drug is covalently linked to one
or more hydrophobic moieties via one or more labile bonds. Said
labile bond, which is preferably hydrolytically labile, may be
silazane or maleamic acid. One of the drugs disclosed (propidium
iodide) contains an aromatic amine, which is reacted with the
respective methylmaleic anhydride to build up a modified drug
containing a dodecyl amine connected to propidium iodide by a
maleamic acid linker. Said maleamic acid linker is branched and
contains a spacer of three carbon atoms between the respective
carbon atoms of the two amido groups. However, the half-life of
said maleimic anilide (aromatic amine containing a drug connected
to the maleamic acid linker) is short in buffered solution at pH of
7.2 (6.1 sec), which is explained by the low pKa of the aniline
type nitrogens on propodium iodide.
[0016] C. Hennard et al., J. Med. Chem. 2001, 44, pages 2139-2151
discloses specific linkers based on succinic acid fragments. The
respective spacers contain 2 succinic acid fragments connected by
an ethylene diamine bridge. One linker additionally contains a
methylenedioxy group. By consequence, each linker contains 4 amide
bonds. Each linker is bound on one side to a pyoverdin backbone and
on the other side to a specific quinolone drug by an aliphatic
amide bond. It was found that the linker which additionally
contains the methylenedioxy group is gradually hydrolysable,
whereas the other linker is stable. By consequence, the adducts
(prodrugs, with the hydrolysable spacer arm) are recommended to be
employed as pharmaceuticals for the therapy against Pseudomonas
aeruginosa, since they have a better activity than those compounds
with the stable spacer, wherein the quinolone is directly bound to
the succinic acid fragment of the respective linker.
[0017] WO 2006/138572 discloses conjugates having a degredable
linkage and polymeric reagents useful in preparing such conjugates.
The conjugates contain a polypeptide as an active agent, the
polymeric reagent is based on an aromatic containing moiety, such a
fluorene, being bound via two spacers to two water-soluble
polymers. The spacer comprises, among others, glutaric acid
fragments, which may form amido bonds with the aromatic containing
moiety. The spacer fragment of the polymeric reagent according to
WO 2006/138572 is found to be stable, the degreadable linkage
within the conjugate is a, for example, --O--C(O)--NH-bridge
connecting the polypeptide with the aromatic containing moiety
originating from the polymeric reagent. Said aromatic containing
fragment is, however, part of a non-biologically active linker for
connecting the polymers with the polypeptide (active agent).
[0018] However, linkers based on succinic acid are also known to be
employed in different applications. U.S. Pat. No. 6,455,268
discloses compounds and methods for detecting and assaying enzyme
activity in an intact cellular system. Said compounds contain a
linking moiety for connecting a fluorescent donor moiety with a
fluorescent acceptor moiety. The linker may contain a fragment
based on succinic acid containing an aromatic and an aliphatic
amide bond. The fluorescent acceptor moiety is connected to the
linker via an aromatic amide. However, it is not reported that said
fluorescent acceptor moiety can be employed as a drug. Instead, the
respective amide bond has to be very stable, since said compound
has to be used as a fluorescent substrate for the detection of
enzyme activities in vivo.
[0019] Accordingly, there is a need for alternative carrier-linked
prodrugs, where the linker allows an autocatalytic cleavage to
release a drug with a controlled release rate and in an unmodified
form without remaining residues originating from the linker.
[0020] Thus, an object of the present invention is to provide such
drug linker conjugates, where the linker is covalently attached via
a cleavable bond to a biologically active moiety (representing the
drug after release), and where the linker is further covalently
attached via a permanent bond to a carrier directly or via a spacer
to form the carrier-linked prodrug.
[0021] This object is achieved by a prodrug or a pharmaceutically
acceptable salt thereof comprising a drug linker conjugate D-L,
wherein [0022] D is an aromatic amine containing biologically
active moiety; and [0023] L is a non-biologically active linker
containing [0024] i) a moiety L.sup.1 represented by formula
(I),
[0024] ##STR00001## [0025] wherein the dashed line indicates the
attachment of L.sup.1 to an aromatic amino group of D by forming an
amide bond; [0026] X.sup.1 is C(R.sup.1R.sup.1a) or a cyclic
fragment selected from C.sub.3-7 cycloalkyl, 4 to 7 membered
heterocyclyl, phenyl, naphthyl, indenyl, indanyl, tetralinyl, or 9
to 11 membered heterobicyclyl, [0027] wherein in case X.sup.1 is a
cyclic fragment, said cyclic fragment is incorporated into L.sup.1
via two adjacent ring atoms and the ring atom of X.sup.1, which is
adjacent to the carbon atom of the amide bond, is also a carbon
atom; [0028] X.sup.2 is a chemical bond or selected from
C(R.sup.3R.sup.3a), N(R.sup.3), O,
C(R.sup.3R.sup.3a)--C(R.sup.4R.sup.4a),
C(R.sup.3R.sup.3a)--N(R.sup.4), N(R.sup.3)--C(R.sup.4R.sup.4a),
C(R.sup.3R.sup.3a)--O, or O--C(R.sup.3R.sup.3a), [0029] wherein in
case X.sup.1 is a cyclic fragment, X.sup.2 is a chemical bond,
C(R.sup.3R.sup.3a), N(R.sup.3) or O; [0030] optionally, in case
X.sup.1 is a cyclic fragment and X.sup.2 is C(R.sup.3R.sup.3a), the
order of the X.sup.1 fragment and the X.sup.2 fragment within
L.sup.1 may be changed and the cyclic fragment is incorporated into
L.sup.1 via two adjacent ring atoms; [0031] R.sup.1, R.sup.3 and
R.sup.4 are independently selected from the group consisting of H,
C.sub.1-4 alkyl and --N(R.sup.5R.sup.5a); [0032] R.sup.1a, R.sup.2,
R.sup.3a, R.sup.4a and R.sup.5a are independently selected from the
group consisting of H, and C.sub.1-4 alkyl; [0033] R.sup.5 is
C(O)R.sup.6; [0034] R.sup.6 is C.sub.1-4 alkyl; [0035] optionally,
one of the pairs R.sup.1a/R.sup.4a, R.sup.3a/R.sup.4a or
R.sup.1a/R.sup.3a form a chemical bond; and [0036] ii) a moiety
L.sup.2, which is a chemical bond or a spacer, and L.sup.2 is bound
to a polymeric carrier group Z, [0037] wherein L.sup.1 is
substituted with one to four L.sup.2 moieties, provided that the
hydrogen marked with the asterisk in formula (I) is not replaced by
L.sup.2; [0038] optionally, L is further substituted.
[0039] It was surprisingly found that prodrugs comprising a drug
linker conjugate D-L containing a linker moiety L.sup.1, as defined
above, exhibit therapeutically useful autohydrolysis (autocatalytic
cleavage) if linked to an aniline derivative or an other type of
aromatic amine through an anilide bond or an amide bond,
respectively. This finding is strongly influenced by the chemical
nature of said linker moiety L.sup.1, which has a first (aliphatic)
amide bond (due to the substituent R.sup.2) and (under the
perspective of the uncleaved drug linker conjugative D-L) a second
(aromatic) amide bond. Between the two amide bonds there is a
spacer (X.sup.2-X.sup.1-fragment), which keeps the respective
carbonyl atoms of the two amido groups (of L.sup.1) in .beta. or
.gamma.-position to each other.
[0040] By consequence, the prodrugs according to the present
invention show the beneficial effect of a controlled release rate
in respect of the (cleaved) drug D-H. Preferably, a sustained
release rate can be obtained. Sustained release (rate) means that
the administration intervals of the respective prodrug are
expanded. Instead of a commonly one or three times daily dosage
form, a (for example) once weekly dosage form (or even longer
administration intervals) can be applied to a person in need
thereof.
[0041] The prodrug according to the present invention show
excellent in vivo/in vitro correlation of linker cleavage, a high
degree of enzyme independence and show a higher stability at lower
pH (pH dependent cleavage).
[0042] Within the present invention the terms are used having the
meaning as follows.
[0043] A strong in vivo/in vitro correlation is observed, if the
release kinetics exhibited by a carrier-linked prodrug conjugate
according to the invention in vivo (plasma levels of free drug) has
a half-life that is not smaller than half the value exhibited by
the same carrier-linked prodrug conjugate in aqueous buffer of pH
7.4 at 37.degree. C. It is understood that in the case of soluble
carriers, release kinetics may be recorded as a hydrolysis
kinetics.
[0044] "Aromatic amine containing biologically active moiety D"
means the part (moiety or fragment) of the drug linker conjugate
D-L, which results after cleavage in a drug D-H (active agent) of
(known) biological activity. In addition, the subterm "aromatic
amine containing" means that the respective moiety D and
analogously the corresponding drug D-H contain at least one
aromatic fragment, which is substituted with at least one amino
group. The terms "drug" and "biologically active moiety" are used
synonymously.
[0045] The amino substituent of the aromatic fragment of D forms
together with the carbonyl-fragment (--C(O)--) on the right hand
side of L.sup.1 (as depicted in formula (I)) an amide bond within
the drug linker conjugate D-L. By consequence, the two parts D and
L of the drug linker conjugate D-L are connected (chemically bound)
by an amide fragment of the general structure
Y.sup.1--C(O)--N(R)--Y.sup.2. Y.sup.1 indicates the remaining parts
of the moiety L.sup.1 and Y.sup.2 indicates the aromatic fragment
of D. R is a substituent such as C.sub.1-4 alkyl or preferably
hydrogen. For example, said amide bond is indicated within formula
(I) by the dashed line added diagonally on this bond.
[0046] "Non-biologically active linker" means a linker which does
not show the pharmacological effects of the drug (D-H) derived from
the biologically active moiety.
[0047] As indicated above, the X.sup.1-fragment of the moiety
L.sup.1 represented by formula (I) may also be a cyclic fragment
such as C.sub.3-7 cycloalkyl, phenyl or indanyl. In case X.sup.1 is
such a cyclic fragment, the respective cyclic fragment is
incorporated into L.sup.1 via two adjacent ring atoms (of said
cyclic fragment). For example, if X.sup.1 is phenyl, the phenyl
fragment of L.sup.1 is bound to the X.sup.2 fragment of L.sup.1 via
a first (phenyl) ring atom being in .alpha.-position (adjacent) to
a second (phenyl) ring atom, which itself is bound to the carbon
atom of the carbonyl-fragment on the right hand side of L.sup.1
according to formula (I) (the carbonyl fragment which forms
together with the aromatic amino group of D an amide bond).
[0048] "Alkyl" means a straight-chain or branched carbon chain
(unsubstituted alkyl). Optionally, each hydrogen of an alkyl carbon
may be replaced by a substituent.
[0049] "C.sub.1-4 alkyl" means an alkyl chain having 1 to 4 carbon
atoms (unsubstituted C.sub.1-4 alkyl), e.g. if present at the end
of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl,
isobutyl, sec-butyl tert-butyl, or e.g. --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH.sub.2--CH.sub.2--CH.sub.2--, --CH(C.sub.2H.sub.5)--,
--C(CH.sub.3).sub.2--, when two moieties of a molecule are linked
by the alkyl group. Optionally, each hydrogen of a C.sub.1-4 alkyl
carbon may be replaced by a substituent. Accordingly, "C.sub.1-50
alkyl" means an alkyl chain having 1 to 50 carbon atoms.
[0050] "C.sub.2-50 alkenyl" means a branched or unbranched alkenyl
chain having 2 to 50 carbon atoms (unsubstituted C.sub.2-50
alkenyl), e.g. if present at the end of a molecule:
--CH.dbd.CH.sub.2, --CH.dbd.CH--CH.sub.3,
--CH.sub.2--CH.dbd.CH.sub.2, --CH.dbd.CH--CH.sub.2--CH.sub.3,
--CH.dbd.CH--CH.dbd.CH.sub.2, or e.g. --CH.dbd.CH--, when two
moieties of a molecule are linked by the alkenyl group. Optionally,
each hydrogen of a C.sub.2-50 alkenyl carbon may be replaced by a
substituent as further specified. Accordingly, the term "alkenyl"
relates to a carbon chain with at least one carbon carbon double
bond. Optionally, one or more triple bonds may occur.
[0051] "C.sub.2-50 alkynyl" means a branched or unbranched alkynyl
chain having 2 to 50 carbon atoms (unsubstituted C.sub.2-50
alkynyl), e.g. if present at the end of a molecule: --C.ident.CH,
--CH.sub.2--C.ident.CH, CH.sub.2--CH.sub.2--C.ident.CH,
CH.sub.2--C.ident.C--CH.sub.3, or e.g. --C.ident.C-- when two
moieties of a molecule are linked by the alkynyl group. Optionally,
each hydrogen of a C.sub.2-50 alkynyl carbon may be replaced by a
substituent as further specified. Accordingly, the term "alkynyl"
relates to a carbon chain with at lest one carbon carbon triple
bond. Optionally, one or more double bonds may occur.
[0052] "C.sub.3-7 cycloalkyl" or "C.sub.3-7 cycloalkyl ring" means
a cyclic alkyl chain having 3 to 7 carbon atoms, which may have
carbon-carbon double bonds being at least partially saturated
(unsubstituted C.sub.3-7 cycloalkyl), e.g. cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Optionally,
each hydrogen of a cycloalkyl carbon may be replaced by a
substituent. The term "C.sub.3-7 cycloalkyl" or "C.sub.3-7
cycloalkyl ring" also includes bridged bicycles like norbonane
(norbonanyl) or norbonene (norbonenyl). Accordingly, "C.sub.3-5
cycloalkyl" means a cycloalkyl having 3 to 5 carbon atoms.
[0053] "Halogen" means fluoro, chloro, bromo or iodo. It is
generally preferred that halogen is fluoro or chloro.
[0054] "4 to 7 membered heterocyclyl" or "4 to 7 membered
heterocycle" means a ring with 4, 5, 6 or 7 ring atoms that may
contain up to the maximum number of double bonds (aromatic or
non-aromatic ring which is fully, partially or un-saturated)
wherein at least one ring atom up to 4 ring atoms are replaced by a
heteroatom selected from the group consisting of sulfur (including
--S(O)--, --S(O).sub.2--), oxygen and nitrogen (including
.dbd.N(O)--) and wherein the ring is linked to the rest of the
molecule via a carbon or nitrogen atom (unsubstituted 4 to 7
membered heterocyclyl). For the sake of completeness it is
indicated that, for example, in case X.sup.1 is 4 to 7 membered
heterocyclyl, the respective additional requirements of X.sup.1
have to be considered as well. This means that in this case the
respective 4 to 7 membered heterocyclyl is incorporated into
L.sup.1 via two adjacent ring atoms and the ring atom of said 4 to
7 membered heterocyclyl, which is adjacent to the carbon atom of
the amide bond, is also a carbon atom.
[0055] Examples for a 4 to 7 membered heterocycles are azetidine,
oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole,
imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole,
isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline,
thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene,
pyrrolidine, imidazolidine, pyrazolidine, oxazolidine,
isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine,
sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine,
pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine,
morpholine, tetrazole, triazole, triazolidine, tetrazolidine,
diazepane, azepine or homopiperazine. Optionally, each hydrogen of
a 4 to 7 membered heterocyclyl may be replaced by a
substituent.
[0056] "9 to 11 membered heterobicyclyl" or "9 to 11 membered
heterobicycle" means a heterocyclic system of two rings with 9 to
11 ring atoms, where at least one ring atom is shared by both rings
and that may contain up to the maximum number of double bonds
(aromatic or non-aromatic ring which is fully, partially or
un-saturated) wherein at least one ring atom up to 6 ring atoms are
replaced by a heteroatom selected from the group consisting of
sulfur (including --S(O)--, --S(O).sub.2--), oxygen and nitrogen
(including .dbd.N(O)--) and wherein the ring is linked to the rest
of the molecule via a carbon or nitrogen atom (unsubstituted 9 to
11 membered heterobicyclyl). For the sake of completeness it is
indicated that, for example, in case X.sup.1 is 9 to 11 membered
heterobicyclyl, the respective additional requirements of X.sup.1
have to be considered as well. This means that in this case the
respective 9 to 11 membered heterobicyclyl is incorporated into
L.sup.1 via two adjacent ring atoms and the ring atom of said 9 to
11 membered heterobicyclyl, which is adjacent to the carbon atom of
the amide bond, is also a carbon atom.
[0057] Examples for a 9 to 11 membered heterobicycle are indole,
indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole,
benzothiazole, benzisothiazole, benzimidazole, benzimidazoline,
quinoline, quinazoline, dihydroquinazoline, quinoline,
dihydroquinoline, tetrahydroquinoline, decahydroquinoline,
isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline,
dihydroisoquinoline, benzazepine, purine or pteridine. The term 9
to 11 membered heterobicycle also includes spiro structures of two
rings like 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles
like 8-aza-bicyclo[3.2.1]octane. Optionally, each hydrogen of a 9
to 11 membered heterobicyclyl may be replaced by a substituent.
[0058] In case the prodrugs according to the present invention
contain one or more acidic or basic groups, the invention also
comprises their corresponding pharmaceutically or toxicologically
acceptable salts, in particular their pharmaceutically utilizable
salts. Thus, the prodrugs which contain acidic groups can be used
according to the invention, for example, as alkali metal salts,
alkaline earth metal salts or as ammonium salts. More precise
examples of such salts include sodium salts, potassium salts,
calcium salts, magnesium salts or salts with ammonia or organic
amines such as, for example, ethylamine, ethanolamine,
triethanolamine or amino acids. Prodrugs which contain one or more
basic groups, i.e. groups which can be protonated, can be present
and can be used according to the invention in the form of their
addition salts with inorganic or organic acids. Examples for
suitable acids include hydrogen chloride, hydrogen bromide,
phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid,
p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid,
acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic
acid, formic acid, propionic acid, pivalic acid, diethylacetic
acid, malonic acid, succinic acid, pimelic acid, fumaric acid,
maleic acid, malic acid, sulfaminic acid, phenylpropionic acid,
gluconic acid, ascorbic acid, isonicotinic acid, citric acid,
adipic acid, and other acids known to the person skilled in the
art. If the prodrugs simultaneously contain acidic and basic groups
in the molecule, the invention also includes, in addition to the
salt forms mentioned, inner salts or betaines (zwitterions). The
respective salts of the prodrugs of the present invention can be
obtained by customary methods which are known to the person skilled
in the art like, for example by contacting these with an organic or
inorganic acid or base in a solvent or dispersant, or by anion
exchange or cation exchange with other salts. The present invention
also includes all salts of the prodrugs which, owing to low
physiological compatibility, are not directly suitable for use in
pharmaceuticals but which can be used, for example, as
intermediates for chemical reactions or for the preparation of
pharmaceutically acceptable salts.
[0059] The term "pharmaceutically acceptable" means approved by a
regulatory agency such as the EMEA (Europe) and/or the FDA (US)
and/or any other national regulatory agency for use in animals,
preferably in humans.
[0060] "Pharmaceutical composition" means a composition containing
one or more active ingredients (for example a drug or a prodrug),
and one or more inert ingredients, as well as any product which
results, directly or indirectly, from combination, complexation or
aggregation of any two or more of the ingredients, or from
dissociation of one or more of the ingredients, or from other types
of reactions or interactions of one or more of the ingredients.
Accordingly, the pharmaceutical compositions of the present
invention encompass any composition made by admixing a prodrug of
the present invention and a pharmaceutically acceptable excipient.
The term "excipient" refers to a diluent, adjuvant, or vehicle with
which the therapeutic is administered. Such pharmaceutical
excipient can be sterile liquids, such as water and oils, including
those of petroleum, animal, vegetable or synthetic origin,
including but not limited to peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water is a preferred excipient when the
pharmaceutical composition is administered orally. Saline and
aqueous dextrose are preferred excipients when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions are preferably employed as
liquid excipients for injectable solutions. Suitable pharmaceutical
excipients include starch, glucose, lactose, sucrose, mannitol,
trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, pH buffering agents, like, for example,
acetate, succinate, tris, carbonate, phosphate, HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES
(2-(N-morpholino)ethanesulfonic acid), or can contain detergents,
like Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids
like, for example, glycine, lysine, or histidine. These
compositions can take the form of solutions, suspensions,
emulsions, tablets, pills, capsules, powders, sustained-release
formulations and the like. The composition can be formulated as a
suppository, with traditional binders and excipients such as
triglycerides. Oral formulation can include standard excipients
such as pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical excipients are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the therapeutic (drug or active ingredient), preferably in
purified form, together with a suitable amount of excipient so as
to provide the form for proper administration to the patient. The
formulation should suit the mode of administration.
[0061] The term "reagent" refers to an intermediate or starting
material used in the assembly process leading to a prodrug of the
present invention.
[0062] The term "chemical functional group" refers to carboxylic
acid and activated derivatives, amino, maleimide, thiol and
derivatives, sulfonic acid and derivatives, carbonate and
derivatives, carbamate and derivatives, hydroxyl, aldehyde, ketone,
hydrazine, isocyanate, isothiocyanate, phosphoric acid and
derivatives, phosphonic acid and derivatives, haloacetyl, alkyl
halides, acryloyl and other alpha-beta unsaturated michael
acceptors, arylating agents like aryl fluorides, hydroxylamine,
disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone,
diazoalkanes, diazoacetyl compounds, oxirane, and aziridine.
[0063] If a chemical functional group is coupled to another
chemical functional group, the resulting chemical structure is
referred to as "linkage". For example, the reaction of an amine
group with a carboxyl group results in an amide linkage.
[0064] "Reactive functional groups" are chemical functional groups
of the backbone moiety, which are connected to the hyperbranched
moiety.
[0065] "Functional group" is the collective term used for "reactive
functional group", "degradable interconnected functional group", or
"conjugate functional group".
[0066] A "degradable interconnected functional group" is a linkage
comprising a biodegradable bond which on one side is connected to a
spacer moiety connected to a backbone moiety and on the other side
is connected to the crosslinking moiety. The terms "degradable
interconnected functional group", "biodegradable interconnected
functional group", "interconnected biodegradable functional group"
and "interconnected functional group" are used synonymously.
[0067] The terms "blocking group" or "capping group" are used
synonymously and refer to moieties which are irreversibly connected
to reactive functional groups to render them incapable of reacting
with for example chemical functional groups.
[0068] The terms "protecting group" or "protective group" refers to
a moiety which is reversibly connected to reactive functional
groups to render them incapable of reacting with for example other
chemical functional groups.
[0069] The term "interconnectable functional group" refers to
chemical functional groups, which participate in a radical
polymerization reaction and are part of the crosslinker reagent or
the backbone reagent.
[0070] The term "polymerizable functional group" refers to chemical
functional groups, which participate in a ligation-type
polymerization reaction and are part of the crosslinker reagent and
the backbone reagent.
[0071] A backbone moiety may comprise a spacer moiety which at one
end is connected to the backbone moiety and on the other side to
the crosslinking moiety.
[0072] The term "derivatives" refers to chemical functional groups
suitably substituted with protecting and/or activation groups or to
activated forms of a corresponding chemical functional group which
are known to the person skilled in the art. For example, activated
forms of carboxyl groups include but are not limited to active
esters, such as succinimidyl ester, benzotriazyl ester, nitrophenyl
ester, pentafluorophenyl ester, azabenzotriazyl ester, acyl
halogenides, mixed or symmetrical anhydrides, acyl imidazole.
[0073] The term "non-enzymatically cleavable linker" refers to
linkers that are hydrolytically degradable under physiological
conditions without enzymatic activity.
[0074] "Non-biologically active linker" means a linker which does
not show the pharmacological effects of the drug (D-H) derived from
the biologically active moiety.
[0075] The terms "spacer", "spacer group", "spacer molecule", and
"spacer moiety" are used interchangeably and if used to describe a
moiety present in the hydrogel carrier of the invention, refer to
any moiety suitable for connecting two moieties, such as C1-50
alkyl, C2-50 alkenyl or C2-50 alkinyl, which fragment is optionally
interrupted by one or more groups selected from --NH--, --N(C1-4
alkyl)-, --O--, --S--, --C(O)--, --C(O)NH--, --C(O)N(C1-4 alkyl)-,
--O--C(O)--, --S(O)--, --S(O)2-, 4 to 7 membered heterocyclyl,
phenyl or naphthyl.
[0076] The terms "terminal", "terminus" or "distal end" refer to
the position of a functional group or linkage within a molecule or
moiety, whereby such functional group may be a chemical functional
group and the linkage may be a degradable or permanent linkage,
characterized by being located adjacent to or within a linkage
between two moieties or at the end of an oligomeric or polymeric
chain.
[0077] The phrases "in bound form" or "moiety" refer to
sub-structures which are part of a larger molecule. The phrase "in
bound form" is used to simplify reference to moieties by naming or
listing reagents, starting materials or hypothetical starting
materials well known in the art, and whereby "in bound form" means
that for example one or more hydrogen radicals (--H), or one or
more activating or protecting groups present in the reagents or
starting materials are not present in the moiety.
[0078] It is understood that all reagents and moieties comprising
polymeric moieties refer to macromolecular entities known to
exhibit variabilities with respect to molecular weight, chain
lengths or degree of polymerization, or the number of functional
groups. Structures shown for backbone reagents, backbone moieties,
crosslinker reagents, and crosslinker moieties are thus only
representative examples.
[0079] A reagent or moiety may be linear or branched. If the
reagent or moiety has two terminal groups, it is referred to as a
linear reagent or moiety. If the reagent or moiety has more than
two terminal groups, it is considered to be a branched or
multi-functional reagent or moiety.
[0080] The term "poly(ethylene glycol) based polymeric chain" or
"PEG based chain" refers to an oligo- or polymeric molecular
chain.
[0081] Preferably, such poly(ethylene glycol) based polymeric chain
is connected to a branching core, it is a linear poly(ethylene
glycol) chain, of which one terminus is connected to the branching
core and the other to a hyperbranched dendritic moiety. It is
understood that a PEG-based chain may be terminated or interrupted
by alkyl or aryl groups optionally substituted with heteroatoms and
chemical functional groups.
[0082] If the term "poly(ethylene glycol) based polymeric chain" is
used in reference to a crosslinker reagent, it refers to a
crosslinker moiety or chain comprising at least 20 weight %
ethylene glycol moieties.
[0083] In the following, the present invention is explained in more
detail.
[0084] D is an aromatic amine containing biologically active
moiety. D may be any aromatic amine containing biologically active
moiety known to a person skilled in the art, which is derived from
the corresponding biologically active drug D-H obtained after
cleavage of D from the drug linker conjugate D-L (D-His the drug or
active agent obtained after release from D-L). As indicated above,
D contains at least one aromatic fragment, which is substituted
with at least one amino group and said amino group is in turn
connected to the moiety L.sup.1 by forming an amide bond.
[0085] The amide bond may be attached to any carbon atom of
suitable substructures of D, including, but not limited to,
thiophenes, pyrroles, imidazoles, pyrazoles, oxazoles, isoxazoles,
thiazoles, isothiazoles, thiadiazoles, thiadiazolines, pyrans,
pyridazines, pyrazines, pyrimidines and tetrazoles.
[0086] D may contain further substituents--besides at least one
aromatic amino group--such as alkyl or halogen. The term "aromatic"
(aromatic fragment) means any aromatic fragment known to a person
skilled in the art such as aryl (for example, phenyl or naphthyl)
or heteroaryl (for example, aromatic 4 to 7 membered heterocyclyl
or aromatic 9 to 11 membered heterobicyclyl). The aromatic fragment
comprises mono-, bi- or polycyclic fragments. In case of bi- or
polycyclic fragments it is sufficient that only one of said cycles
is aromatic. D may contain two or more further aromatic fragments
as defined before, which are bound to the first aromatic fragment,
which is substituted with at least one amino group, either directly
by a chemical bound or by a spacer. Said two or more additional
aromatic fragments may also contain at least one amino group. In
the following, D is defined by the corresponding biologically
active drug D-H.
[0087] D-His preferably selected from the group consisting of
Abacavir, Acadesine, Acediasulfone, Aciclovir, Actimid,
Actinomycin, Adefovir, Aditeren, Afloqualone, Aztreonam, Adefovir
Dipivoxil, Adenine, Adenosine, Adenosine monophosphate, Adenosine
triphosphate, Alfuzosin, Alpiropride, Ambasilide, Ambucaine,
Ameltolide, Amethopterin, Amicycline, Amidapsone, Amiloride,
Aminoacridine, Aminoantipyrine, Aminobenzoate, 6-Aminoflavone,
17-Aminogeldanamycin, Aminogenistein, Aminoglutethimide,
Aminohippurate, 3'-Amino-4'-methoxyflavone, Aminonimetazepam,
Aminopotentidine, Amphenidone, N-(p-Aminophenethyl)spiroperidol,
2-Amino-6(5H)-phenanthridinone, Amiphenosine, Aminophenylalanine,
Aminopterin, Aminopurvalanol A, Amfenac, Amiphenazole, Amphotalide,
Aminoisatin, Aminosalicylic Acid, Amifampridine, Amisulpride,
Amlexanox, Amonafide, Amprenavir, Amrinone, Amthamine, Anileridine,
Apraclonidine, Ascensil, Atolide, Azabon, Azacitidine, Azepexole,
Aztreonam, Basedol, Benzocaine, Batanopride, Betoxycaine,
Bleomycin, Bromfenac, Bromobuterol, Bromopride, Carbutamide,
Carumonam, Candicidin, Cefepime, Cefcapene pivoxil, Cefdaloxime,
Cefdinir, Cefditoren, Cefempidone, Cefetamet, Cefepime, Cefetecol,
Cefixime, Cefmatilen, Cefmenoxime, Cefodizime, Cefoselis,
Cefotaxime, Cefotiam, Ceftiolene, Ceftioxide, Cefpodoxime,
Cefquinome, Cefrom, Ceftazidime, Cefteram, Ceftibuten, Ceftiofur,
Ceftizoxime, Ceftriaxone, Cefuzonam, Cisapride, Clenproperol,
Chloroprocaine, Cidofovir, Cisapride, Cladribine, Clafanone,
Claforan, Clebopride, Clenbuterol, Clofarabine, Clorsulon,
Cycloclenbuterol, Cytarabine, Cytidoline, Dactinomycin,
Daniquidone, Dactinomycin, Dapsone, Daptomycin, Daraprim,
Darunavir, Dazopride, Decitabine, Declopramide, Diaminoacridine,
Dichlorophenarsine, Dimethocaine, 10'-Demethoxystreptonigrin,
2,7-Dimethylproflavine, Dinalin, Dobupride, Doxazosin, Draflazine,
Emtricitabine, Entecavir, Ethacridine, Etanterol, Etoxazene,
Famciclovir, Fepratset, (.+-.)-FLA 668, Flucytosine, Fludarabine,
Folic Acid, Fosamprenavir, Ganciclovir, Gemcitabine, Gloximonam,
GSK 3B Inhibitor XII, Glybuthiazol, Hydroxymethylclenbuterol,
Hydroxyprocaine, Imiquimod, Indanocine, Iomeglamic acid, Iramine,
Isobutamben, Isoritmon, Ketoclenbuterol, Lamivudine, Lamotrigine,
Lavendamycin, Lenalidomide, Leucinocaine, Leucovorin, Lintopride,
Lisadimate, Mabuterol, Medeyol, Mesalazine, Metabutethamine,
Metabutoxycaine, Metahexamide, Methyl anthranilate, Methotrexate,
Metoclopramide, Minoxidil, Mirabegron, Mitomycin, Mocetinostat,
Monocain, Mosapride, NADH, Mutamycin, Naepaine, Naminterol,
Nelarabine, Nepafenac, Nerisopam, Nitrine, Nomifensine,
Norcisapride, Olamufloxacin, Orthocaine, Oxybuprocaine, Oximonam,
Pancopride, Parsalmide, Pathocidine, Pasdrazide, Pemetrexed,
Penciclovir, Phenazone, Phenazopyridine, Phenyl-PAS-Tebamin,
Picumeterol, Pirazmonam, Porfiromycin, Pramipexole, Prazosin,
Piridocaine, Procainamide, Procaine, Proflavine,
N-Propionylprocainamide, Proparacaine, Propoxycaine, Prucalopride,
Pyrimethamine, Questiomycin, Renoquid, Renzapride, Retigabine,
Riluzole, Rufocromomycin, S-Adenosylmethionine, Silver
sulfadiazine, Sparfloxacin, Stearylsulfamide, Streptonigrin,
Succisulfone, Sulamserod, Sulfabromomethazine, Sulfacetamide,
Sulfaclozine, Sulfaclorazole, Sulfachlorpyridazine,
Sulfachrysoidine, Sulfaclomide, Sulfacytine, Sulfadiasulfone,
Sulfadimethoxine, Sulfadimidine, Sulfadicramide, Sulfadiazine,
Sulfadoxine, Sulfaguanidine, Sulfaguanole, Sulfalene,
Sulfamerazine, Sulfamethazine, Sulfanilamidoimidazole,
Sulfanilylglycine, N-Sulfanilylnorfloxacin, Sulfathiadiazole,
Sulfamethizole, Sulfamethoxazole, Sulfametopyrazine, Sulfapyrazole,
Sulfamethoxydiazine, Sulfasymazine, Sulfatrozole, Sulfatroxazole,
Sulfamethoxypyridazine, Sulfametomidine, Sulfametrole,
Sulfamonomethoxine, Sulfanilamide, Sulfaperin, Sulfaphenazole,
Sulfaproxyline, Sulfapyridine, Sulfisomidine, Sulfasomizole,
Sulfisoxazole, Suprax, Tacedinaline, Tacrine, Talampanel,
Talipexole, Tenofovir, Terazosin, Tetrahydrobiopterin,
Tetrahydrofolic acid, Thiamine, Thiazosulfone, Thioguanine,
Tigemonam, Timirdine, Trimethoprim, Triamterene, Trimethoprim,
Trimetrexate, Tritoqualine, Valaciclovir, Valganciclovir,
Veradoline, Vidarabine, Zalcitabine, and Zoxazolamine.
[0088] Most preferably, D-His pramipexole.
[0089] In one embodiment of the present invention, D (or D-H,
respectively) does not contain a fluorenyl fragment substituted
with the aromatic amino group. Aromatic amino group means that said
group is attached in the drug linker conjugate D-L to the moiety
L.sup.1 by forming the amide bond.
[0090] The non-biologically active linker L contains a moiety
L.sup.1 represented by formula (I) as depicted and defined above.
Preferably, the moiety L.sup.1 is defined as follows. [0091]
X.sup.1 is C(R.sup.1R.sup.1a), cyclohexyl, phenyl, pyridinyl,
norbonenyl, furanyl, pyrrolyl or thienyl, [0092] wherein in case
X.sup.1 is a cyclic fragment, said cyclic fragment is incorporated
into L.sup.1 via two adjacent ring atoms; [0093] X.sup.2 is a
chemical bond or selected from C(R.sup.3R.sup.3a), N(R.sup.3), O,
C(R.sup.3R.sup.3a)--O or C(R.sup.3R.sup.3a)--C(R.sup.4R.sup.4a);
[0094] R.sup.1, R.sup.3 and R.sup.4 are independently selected from
H, C.sub.1-4 alkyl or --N(R.sup.5R.sup.5a); [0095] R.sup.1a,
R.sup.3a, R.sup.4a and R.sup.5a are independently selected from H
or C.sub.1-4 alkyl; [0096] R.sup.2 is C.sub.1-4 alkyl; [0097]
R.sup.5 is C(O)R.sup.6; [0098] R.sup.6 is C.sub.1-4 alkyl;
[0099] More preferably, the moiety L.sup.1 is selected from
##STR00002## ##STR00003## ##STR00004## ##STR00005##
wherein
R.sup.5 is C(O)R.sup.6;
[0100] R.sup.1, R.sup.1a, R.sup.2, R.sup.3 and R.sup.6 are
independently from each other C.sub.1-4 alkyl; and L.sup.1 is
substituted with one L.sup.2 moiety, preferably R.sup.2 is
substituted with one L.sup.2 moiety.
[0101] In one embodiment of the present invention, the moiety
L.sup.1 is even more preferably
##STR00006##
wherein
R.sup.5 is C(O)R.sup.6;
[0102] R.sup.6 is C.sub.1-4 alkyl; and R.sup.2 is substituted with
one L.sup.2.
[0103] In another embodiment of the present invention, the moiety
L.sup.1 is even more preferably
##STR00007##
wherein R.sup.1, R.sup.1a, R.sup.2, and R.sup.3 are independently
from each other selected from H or C.sub.1-4 alkyl; and R.sup.2 is
substituted with one L.sup.2 moiety.
[0104] L also contains a moiety L.sup.2, which is a chemical bond
or a spacer. L.sup.2 is bound to a carrier group Z. L.sup.1 is
substituted with 1 to 4 L.sup.2 moieties, provided that the
hydrogen marked with the asterisk in formula (I) is not replaced by
L.sup.2. In case more than one L.sup.2 moiety is present, each
L.sup.2 and, by consequence, each Z can be selected independently.
In general, L.sup.2 can be attached to L.sup.1 at any position
apart from the replacement of the hydrogen marked with an asterisk
in formula (I). The attachment of the respective L.sup.2 moiety
occurs by replacing one hydrogen according to the definitions of
X.sup.1, X.sup.2, R.sup.1 to R.sup.5 and R.sup.1a to R.sup.5a.
Preferably, L.sup.1 is substituted with one L.sup.2 moiety. More
preferably, R.sup.2 is substituted with one L.sup.2 moiety (the
substitution occurs at the R.sup.2 fragment of the moiety L.sup.1).
In particular, R.sup.2 is substituted with L.sup.2 at the terminal
carbon atom, if R.sup.2 is C.sub.1-4 alkyl, preferably butyl.
[0105] In another embodiment, the moiety L.sup.2 may entirely
replace R.sup.2 within formula (I) or the preferred definitions
thereof, respectively.
[0106] In case L.sup.2 is a spacer, any spacer known to a person
skilled in the art for connecting a moiety L.sup.1 as represented
by formula (I) to a carrier can be used. Preferably, the spacer is
a fragment selected from C.sub.1-50 alkyl, C.sub.2-50 alkenyl or
C.sub.2-50 alkinyl, which fragment is optionally interrupted by one
or more groups selected from --NH--, --N(C.sub.1-4 alkyl)-, --O--,
--S--, --C(O)--, --C(O)NH--, --C(O)N(C.sub.1-4 alkyl)-,
--O--C(O)--, --S(O)--, --S(O).sub.2--, 4 to 7 membered
heterocyclyl, phenyl or naphthyl.
[0107] More preferably, the spacer is a fragment selected from
C.sub.1-50 alkyl, C.sub.2-50 alkenyl or C.sub.2-50 alkinyl, which
fragment is optionally interrupted by one or more groups selected
from --NH--, --N(C.sub.1-4 alkyl)-, --O--, --S--, --C(O)--,
--C(O)NH--, --C(O)N(C.sub.1-4 alkyl)-, --O--C(O)--, --S(O)--,
--S(O).sub.2--, 4 to 7 membered heterocyclyl, phenyl or naphthyl,
provided that the spacer does not contain a nitrogen atom being in
.beta.- or .gamma.-position to the amino group containing the
hydrogen marked with the asterisk in formula (I), in case the
spacer is bound to R.sup.2.
[0108] The term "interrupted" means that between two carbon atoms
of the spacer or at the end of the carbon chain between the
respective carbon atom and the hydrogen atom a group (as defined
above) is inserted.
[0109] Even more preferably, the spacer is a C.sub.1-20 alkyl being
bound to R.sup.2 and which C.sub.1-20 alkyl is optionally
interrupted by one or more groups selected from --NH--,
--N(C.sub.1-4 alkyl)-, --O--, --S--, --C(O)--, --C(O)NH--,
--C(O)N(C.sub.1-4 alkyl)-, --O--C(O)--, --S(O)--, --S(O).sub.2--, 4
to 7 membered heterocyclyl, phenyl or naphthyl, provided that the
spacer does not contain a nitrogen atom being in .beta.- or
.gamma.-position to the amino group containing the hydrogen marked
with the asterisk in formula (I).
[0110] According to another embodiment of the present invention, it
is preferred that L.sup.2 has a molecular weight in the range of
from 14 g/mol to 750 g/mol.
[0111] Preferably, L.sup.2 is attached to Z via a terminal group
selected from
##STR00008##
and
##STR00009##
or --CO--NH--, most preferred --CO--NH--.
[0112] In case L.sup.2 has such terminal group it is furthermore
preferred that L.sup.2 has a molecular weight in the range of from
14 g/mol to 500 g/mol calculated without such terminal group.
[0113] In another embodiment of the present invention, the spacer
is preferably a C.sub.1-20 alkyl being bound to R.sup.2 and which
C.sub.1-20 alkyl is optionally interrupted by one or more groups
selected from --NH--, --N(C.sub.1-4 alkyl)-, --O--, --S--,
--C(O)--, --C(O)NH--, --C(O)N(C.sub.1-4 alkyl)-, --O--C(O)--,
--S(O)--, --S(O).sub.2--, 4 to 7 membered heterocyclyl, phenyl or
naphthyl, provided that the spacer does not contain a nitrogen atom
being in .beta.- or .gamma.-position to the nitrogen atom bound to
the hydrogen marked with the asterisk in formula (I) and L.sup.2 is
attached to Z via a terminal group selected from
##STR00010##
and
##STR00011##
or --CO--NH--, most preferred --CO--NH--, whereby L.sup.2 has a
molecular weight in the range of from 14 g/mol to 500 g/mol
calculated without said terminal group.
[0114] In one embodiment of the present invention, the
non-biologically active linker L of the drug linker conjugate D-L
may be optionally substituted further by one or more substituents.
The substitution may occur at the moiety L.sup.1 and/or the moiety
L.sup.2 including the respective preferred definitions of L.sup.1
and/or L.sup.2. In general, any substituent may be used as far as
the cleavage principle is not affected.
[0115] Preferably, one or more further optional substituents are
independently selected from the group consisting of halogen; CN;
COOR.sup.9; OR.sup.9; C(O)R.sup.9; C(O)N(R.sup.9R.sup.9a);
S(O).sub.2N(R.sup.9R.sup.9a); S(O)N(R.sup.9R.sup.9a);
S(O).sub.2R.sup.9; S(O)R.sup.9;
N(R.sup.9)S(O).sub.2N(R.sup.9aR.sup.9b); SR.sup.9;
N(R.sup.9R.sup.9a); NO.sub.2; OC(O)R.sup.9; N(R.sup.9)C(O)R.sup.9a;
N(R.sup.9)S(O).sub.2R.sup.9a; N(R.sup.9)S(O)R.sup.9a;
N(R.sup.9)C(O)OR.sup.9a; N(R.sup.9)C(O)N(R.sup.9aR.sup.9b);
OC(O)N(R.sup.9R.sup.9a); T; C.sub.1-50 alkyl; C.sub.2-50 alkenyl;
or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl; C.sub.2-50
alkenyl; and C.sub.2-50 alkynyl are optionally substituted with one
or more R.sup.10, which are the same or different and wherein
C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are
optionally interrupted by one or more groups selected from the
group consisting of T, --C(O)O--; --O--; --C(O)--;
--C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.1a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0116] R.sup.9, R.sup.9a, R.sup.9b are independently selected from
the group consisting of H; T; and C.sub.1-50 alkyl; C.sub.2-50
alkenyl; or C.sub.2-50 alkynyl, wherein T; C.sub.1-50 alkyl;
C.sub.2-50 alkenyl; and C.sub.2-50 alkynyl are optionally
substituted with one or more R.sup.10, which are the same or
different and wherein C.sub.1-50 alkyl; C.sub.2-50 alkenyl; and
C.sub.2-50 alkynyl are optionally interrupted by one or more groups
selected from the group consisting of T, --C(O)O--; --O--;
--C(O)--; --C(O)N(R.sup.11)--; --S(O).sub.2N(R.sup.11)--;
--S(O)N(R.sup.11)--; --S(O).sub.2--; --S(O)--;
--N(R.sup.11)S(O).sub.2N(R.sup.11a)--; --S--; --N(R.sup.11)--;
--OC(O)R.sup.11; --N(R.sup.11)C(O)--; --N(R.sup.11)S(O).sub.2--;
--N(R.sup.11)S(O)--; --N(R.sup.11)C(O)O--;
--N(R.sup.11)C(O)N(R.sup.1a)--; and
--OC(O)N(R.sup.11R.sup.11a);
[0117] T is selected from the group consisting of phenyl; naphthyl;
indenyl; indanyl; tetralinyl; C.sub.3-10 cycloalkyl; 4 to 7
membered heterocyclyl; or 9 to 11 membered heterobicyclyl, wherein
T is optionally substituted with one or more R.sup.10, which are
the same or different;
[0118] R.sup.10 is halogen; CN; oxo (.dbd.O); COOR.sup.12;
OR.sup.12; C(O)R.sup.12; C(O)N(R.sup.12R.sup.12a);
S(O).sub.2N(R.sup.12R.sup.12a); S(O)N(R.sup.12R.sup.12a);
S(O).sub.2R.sup.12; S(O)R.sup.12;
N(R.sup.12)S(O).sub.2N(R.sup.12aR.sup.12); SR.sup.12;
N(R.sup.12R.sup.12a); NO.sub.2; OC(O)R.sup.12;
N(R.sup.12)C(O)R.sup.12a; N(R.sup.12)S(O).sub.2R.sup.12a;
N(R.sup.12)S(O)R.sup.12a; N(R.sup.12)C(O)OR.sup.12a;
N(R.sup.12)C(O)N(R.sup.12aR.sup.12b); OC(O)N(R.sup.12R.sup.12a); or
C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted
with one or more halogen, which are the same or different;
[0119] R.sup.11, R.sup.11a, R.sup.12, R.sup.12a, R.sup.12b are
independently selected from the group consisting of H; or C.sub.1-6
alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one
or more halogen, which are the same or different.
[0120] The carrier group Z is bound to the moiety L.sup.2. In case
the moiety L.sup.2 is a chemical bond, the carrier group Z is
directly bound to the moiety L.sup.1 without a spacer in-between.
For the sake of completeness, it is indicated that in case L
contains more than one L.sup.2 moieties and by consequence more
than one carrier group Z. The respective carrier groups Z may be
selected independently from each other and independently from the
definition of L.sup.2. For example, in case of two L.sup.2
moieties, one carrier group Z can be directly bound to L.sup.1
(L.sup.2 is a chemical bond) and the second (different) carrier
group Z can be bound to L.sup.1 by a spacer (L.sup.2). The carrier
group Z can be any carrier group known to a person skilled in the
art.
[0121] Suitable carriers are polymers or C.sub.8-18 alkyl
groups.
[0122] Preferably, the carrier group Z is a polymer, more
preferably a polymer with a molecular weight .gtoreq.500 g/mol.
[0123] The term polymer describes a molecule comprised of repeating
structural units connected by chemical bonds in a linear, circular,
branched, crosslinked or dendrimeric way or a combination thereof,
which can be of synthetic or biological origin or a combination of
both.
[0124] Preferred polymers are selected from 2-methacryloyl-oxyethyl
phosphoyl cholins, hydrogels, PEG-based hydrogels, poly(acrylic
acids), poly(acrylates), poly(acrylamides), poly(alkyloxy)
polymers, poly(amides), poly(amidoamines), poly(amino acids),
poly(anhydrides), poly(aspartamides), poly(butyric acids),
poly(glycolic acids), polybutylene terephthalates,
poly(caprolactones), poly(carbonates), poly(cyanoacrylates),
poly(dimethylacrylamides), poly(esters), poly(ethylenes),
poly(ethyleneglycols), poly(ethylene oxides), poly(ethyl
phosphates), poly(ethyloxazolines), poly(glycolic acids),
poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),
poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),
poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),
poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic
acids), poly(methacrylamides), poly(methacrylates),
poly(methyloxazolines), poly(organophosphazenes), poly(ortho
esters), poly(oxazolines), poly(propylene glycols),
poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl
amines), poly(vinylmethylethers), poly(vinylpyrrolidones),
silicones, celluloses, carbomethyl celluloses, hydroxypropyl
methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins,
hyaluronic acids and derivatives, mannans, pectins,
rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl
starches and other carbohydrate-based polymers, xylans, and
copolymers thereof.
[0125] As indicated above, the carrier group Z may be a hydrogel
(as one option for a polymer). Hydrogels to be used are known in
the art. Suitable hydrogels may be used which are described in WO-A
2006/003014 or EP-A 1 625 856. Accordingly, a hydrogel may be
defined as a three-dimensional, hydrophilic or amphiphilic
polymeric network capable of taking up large quantities of water.
The networks are composed of homopolymers or copolymers, are
insoluble due to the presence of covalent chemical or physical
(ionic, hydrophobic interactions, entanglements) crosslinks. The
crosslinks provide the network structure and physical integrity.
Hydrogels exhibit a thermodynamic compatibility with water which
allow them to swell in aqueous media. The chains of the network are
connected in such a fashion that pores exist and that a substantial
fraction of these pores are of dimensions between 1 nm and 1000
nm.
[0126] In a preferred embodiment of the present invention, the
carrier group Z is a biodegradable polyethylene glycol based
water-insoluble hydrogel comprising backbone moieties which are
linked together by hydrolytically degradable bonds. In one
embodiment the water-insoluble hydrogel is further characterized in
that the time period for the complete degradation of the hydrogel
by hydrolysis of the degradable bonds into water-soluble
degradation products comprising one or more backbone moieties is
twice or less than the time period for the release of the first 10
mol-% of backbone moieties based on the total amount of backbone
moieties in the hydrogel.
[0127] The hydrolytically degradable bonds of said hydrogels
preferably are ester bonds. The backbone moieties can be linked
together by crosslinkers.
[0128] Preferably, the covalent attachment formed between the
linker and the carrier is a permanent bond and the carrier is a
hydrogel.
[0129] Preferably, the carrier is a biodegradable polyethylene
glycol (PEG) based water-insoluble hydrogel. The term "PEG based"
as understood herein means that the mass proportion of PEG chains
in the hydrogel is at least 10% by weight, preferably at least 25%,
based on the total weight of the hydrogel. The remainder can be
made up of other spacers and/or oligomers or polymers, such as
oligo- or polylysines.
[0130] Moreover the term "water-insoluble" refers to a swellable
three-dimensionally crosslinked molecular network froming the
hydrogel. The hydrogel if suspended in a large surplus of water or
aqueous buffer of physiological osmolality may take up a
substantial amount of water, e.g. up to 10-fold on a weight per
weight basis, and is therefore swellable but after removing excess
water still retains the physical stability of a gel and a shape.
Such shape may be of any geometry and it is understood that such an
individual hydrogel object is to be considered as a single molecule
consisting of components wherein each component is connected to
each other component through chemical bonds.
[0131] Another aspect of the present invention is a carrier-linked
prodrug comprising a biodegradable hydrogel of the present
invention as carrier, wherein a number of permanent linkages of the
backbone moieties exist with a transient prodrug linker to which a
biologically active moiety is covalently attached.
[0132] The reactive functional groups of a reactive biodegradable
hydrogel or modified reactive biodegradable hydrogel serve as
attachment points for direct linkage through the before mentioned
permanent linkages of drug-linker conjugate. Ideally, the
hydrogel-connected drug-linker conjugates are dispersed
homogeneously throughout the hydrogel according to the invention,
and may or may not be present on the surface of the hydrogel
according to the invention.
[0133] The functional groups may be attached to a linear chain. In
this case, the functional groups may be spaced regularly or
irregularly across the chain, or alternatively, the chain may be
terminated by two dendritic moieties, providing for the total of
functional groups.
[0134] Remaining reactive functional groups which are not connected
to a transient prodrug linker or to a spacer connected to a
transient prodrug linker may be capped with suitable blocking
reagents.
[0135] Preferably, the covalent attachment formed between the
reactive functional groups provided by the backbone moieties and
the prodrug linker are permanent bonds. Suitable functional groups
for attachment of the prodrug linker to the hydrogel according to
the invention include but are not limited to carboxylic acid and
derivatives, carbonate and derivatives, hydroxyl, hydrazine,
hydroxylamine, maleamic acid and derivatives, ketone, amino,
aldehyde, thiol and disulfide.
[0136] According to this invention, the biodegradable hydrogel
according to the invention is composed of backbone moieties
interconnected by hydrolytically degradable bonds.
[0137] In a hydrogel carrying drug-linker conjugates according to
the invention, a backbone moiety is characterized by a number of
functional groups, comprising interconnected biodegradable
functional groups and hydrogel-connected drug-linker conjugates,
and optionally capping groups. This means that a backbone moiety is
characterized by a number of hydrogel-connected drug-linker
conjugates; functional groups, comprising biodegradable
interconnected functional groups; and optionally capping groups.
Preferably, the sum of interconnected biodegradable functional
groups and drug-linker conjugates and capping groups is 16-128,
preferred 20-100, more preferred 24-80 and most preferred
30-60.
[0138] Preferably, the sum of interconnected functional groups and
hydrogel-connected drug-linker conjugates and capping groups of a
backbone moiety is equally divided by the number of PEG-based
polymeric chains extending from the branching core. For instance,
if there are 32 interconnected functional groups and
hydrogel-connected drug-linker conjugates and capping groups, eight
groups may be provided by each of the four PEG-based polymeric
chains extending from the core, preferably by means of dendritic
moieties attached to the terminus of each PEG-based polymeric
chain. Alternatively, four groups may be provided by each of eight
PEG-based polymeric chains extending from the core or two groups by
each of sixteen PEG-based polymeric chains. If the number of
PEG-based polymeric chains extending from the branching core does
not allow for an equal distribution, it is preferred that the
deviation from the mean number of the sum of interconnected
functional groups and hydrogel-connected drug-linker conjugates and
capping groups per PEG-based polymeric chain is kept to a
minimum.
[0139] In such carrier-linked prodrugs according to the invention,
it is desirable that almost all drug release (>90%) has occurred
before a significant amount of release of the backbone moieties
(<10%) has taken place. This can be achieved by adjusting the
carrier-linked prodrug's half-life versus the degradation kinetics
of the hydrogel according to the invention.
[0140] In a hydrogel according to the invention, a backbone moiety
is characterized by a number of functional groups, consisting of
interconnected biodegradable and reactive functional groups.
Preferably, the sum of interconnected biodegradable and reactive
functional groups is 16-128, preferred 20-100, more preferred 24-80
and most preferred 30-60.
[0141] The functional groups may be attached to a linear chain. In
this case, the functional groups may be spaced regularly or
irregularly across the chain, or alternatively, the chain may be
terminated by two dendritic moieties, providing for the total of
functional groups.
[0142] Preferentially, a backbone moiety is characterized by having
a branching core, from which at least three PEG-based polymeric
chains extend. Accordingly, in a preferred aspect of the present
invention the backbone reagent comprises a branching core, from
which at least three PEG-based polymeric chains extend. Such
branching cores may be comprised of poly- or oligoalcohols in bound
form, preferably pentaerythritol, tripentaerythritol,
hexaglycerine, sucrose, sorbitol, fructose, mannitol, glucose,
cellulose, amyloses, starches, hydroxyalkyl starches,
polyvinylalcohols, dextranes, hyualuronans, or branching cores may
be comprised of poly- or oligoamines such as ornithine,
diaminobutyric acid, trilysine, tetralysine, pentalysine,
hexylysine, heptalysine, octalysine, nonalysine, decalysine,
undecalysine, dodecalysine, tridecalysine, tetradecalysine,
pentadecalysine or oligolysines, polyethyleneimines,
polyvinylamines in bound form.
[0143] Preferably, the branching core extends three to sixteen
PEG-based polymeric chains, more preferably four to eight.
Preferred branching cores may be comprised of pentaerythritol,
ornithine, diaminobutyric acid, trilysine, tetralysine,
pentalysine, hexylysine, heptalysine or oligolysine, low-molecular
weight PEI, hexaglycerine, tripentaerythritol in bound form.
Preferably, the branching core extends three to sixteen PEG-based
polymeric chains, more preferably four to eight. Preferably, a
PEG-based polymeric chain is a linear poly(ethylene glycol) chain,
of which one end is connected to the branching core and the other
to a hyperbranched dendritic moiety. It is understood that a
polymeric PEG-based chain may be terminated or interrupted by alkyl
or aryl groups optionally substituted with heteroatoms and chemical
functional groups.
[0144] Preferentially, a backbone moiety is characterized by having
a branching core, from which at least three chains extend. Such
branching cores may be provided by suitably substituted derivatives
of poly- or oligoalcohols, preferably pentaerythritol,
tripentaerythritol, hexaglycerine, sucrose, sorbitol, fructose,
mannitol, glucose, cellulose, amyloses, starches, hydroxyalkyl
starches, polyvinylalcohols, dextranes, hyualuronans, or branching
cores may be provided by suitably substituted derivatives of poly-
or oligoamines such as trilysine, tetralysine, pentalysine,
hexylysine, heptalysine, octalysine, nonalysine, decalysine,
undecalysine, dedecalysine, tridecalysine, tetradecalysine,
pentadecalysine or oligolysines, polyethyleneimines,
polyvinylamines. Preferably, the branching core extends three to
sixteen chains, more preferably four to eight. Preferably, such
chain is a linear polyethylene glycol chain, of which one end is
connected to the branching core and the other to a hyperbranched
dendritic moiety.
[0145] Preferably, a PEG-based polymeric chain is a suitably
substituted polyethylene glycol derivative (PEG based).
[0146] Preferred structures for corresponding PEG-based polymeric
chains extending from a branching core contained in a backbone
moiety are multi-arm PEG derivatives as, for instance, detailed in
the products list of JenKem Technology, USA (accessed by download
from www.jenkemusa.com on Jul. 28, 2009), 4ARM-PEG Derivatives
(pentaerythritol core), 8ARM-PEG Derivatives (hexaglycerin core)
and 8ARM-PEG Derivatives (tripentaerythritol core). Most preferred
are 4arm PEG Amine (pentaerythritol core) and 4arm PEG Carboxyl
(pentaerythritol core), 8arm PEG Amine (hexaglycerin core), 8arm
PEG Carboxyl (hexaglycerin core), 8arm PEG Amine
(tripentaerythritol core) and 8arm PEG Carboxyl (tripentaerythritol
core). Preferred molecular weights for such multi-arm
PEG-derivatives in a backbone moiety are 1 kDa to 20 kDa, more
preferably 1 kDa to 15 kDa and even more preferably 1 kDa to 10
kDa.
[0147] It is understood that the terminal amine groups of the above
mentioned multi-arm molecules are present in bound form in the
backbone moiety to provide further interconnected functional groups
and reactive functional groups of a backbone moiety.
[0148] Preferred branching cores may be provided by suitably
substituted derivatives of pentaerythritol, trilysine, tetralysine,
pentalysine, hexylysine, heptalysine or oligolysine, low-molecular
weight PEI, hexaglycerine, tripentaerythritol. Preferably, a chain
is a suitably substituted polyethylene glycol derivative (PEG
based).
[0149] It is preferred that the sum of interconnected functional
groups and reactive functional groups of a backbone moiety is
equally divided by the number of PEG-based polymeric chains
extending from the branching core. If the number of PEG-based
polymeric chains extending from the branching core does not allow
for an equal distribution, it is preferred that the deviation from
the mean number of the sum of interconnected and reactive
functional groups per PEG-based polymeric chain is kept to a
minimum.
[0150] More preferably, the sum of interconnected and reactive
functional groups of a backbone moiety is equally divided by the
number of PEG-based polymeric chains extending from the branching
core. For instance, if there are 32 interconnected functional
groups and reactive functional groups, eight groups may be provided
by each of the four PEG-based polymeric chains extending from the
core, preferably by means of dendritic moieties attached to the
terminus of each PEG-based polymeric chain. Alternatively, four
groups may be provided by each of eight PEG-based polymeric chains
extending from the core or two groups by each of sixteen PEG-based
polymeric chains.
[0151] Preferably, the sum of interconnecting biodegradable
linkages and permanent linkages connecting backbone moieties to
prodrug-linker and blocking groups is equally divided by the number
of chains extending from the branching core. For instance, if there
are 32 interconnected and biodegradable and permanent linkages,
eight linkages may be provided by each of the four chains extending
from the core, preferably by means of dendritic moieties attached
to the terminus of each chain. Alternatively, four linkages may be
provided by each of eight chains extending from the core or two
groups by each of sixteen chains.
[0152] The sum of interconnected and reactive functional groups of
a backbone moiety is equally divided by the number of chains
extending from the branching core. If the number of chains
extending from the branching core does not allow for an equal
distribution, it is preferred that the deviation from the mean
number of the sum of interconnected and reactive functional groups
per chain is kept to a minimum.
[0153] More preferably, the sum of interconnected and reactive
functional groups of a backbone moiety is equally divided by the
number of chains extending from the branching core. For instance,
if there are 32 interconnected and reactive functional groups,
eight groups may be provided by each of the four chains extending
from the core, preferably by means of dendritic moieties attached
to the terminus of each chain. Alternatively, four groups may be
provided by each of eight chains extending from the core or two
groups by each of sixteen chains.
[0154] Such additional functional groups may be provided by
dendritic moieties. Preferably, each dendritic moiety has a
molecular weight in the range of from 0.4 kDa to 4 kDa, more
preferably 0.4 kDa to 2 kDa. Preferably, each dendritic moiety has
at least 3 branchings and at least 4 reactive functional groups,
and at most 63 branchings and 64 reactive functional groups,
preferred at least 7 branchings and at least 8 reactive functional
groups and at most 31 branchings and 32 reactive functional
groups.
[0155] Examples for such dendritic moieties are comprised of
trilysine, tetralysine, pentalysine, hexylysine, heptalysine,
octalysine, nonalysine, decalysine, undecalysine, dodecalysine,
tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,
heptadecalysine, octadecalysine, nonadecalysine in bound form.
Examples for such preferred dendritic moieties are comprised
oftrilysine, tetralysine, pentalysine, hexylysine, heptalysine in
bound form, most preferred trilysine, pentalysine or heptalysine,
ornithine, diaminobutyric acid in bound form.
[0156] Preferred structures for corresponding chains extending from
a branching core contained in a backbone moiety are multi-arm PEG
derivatives as, for instance, detailed in the products list of
JenKem Technology, USA (accessed by download from www.jenkemusa.com
on Jul. 28, 2009), 4ARM-PEG Derivatives (pentaerythritol core),
8ARM-PEG Derivatives (hexaglycerin core) and 8ARM-PEG Derivatives
(tripentaerythritol core). Most preferred are 4arm PEG Amine
(pentaerythritol core) and 4arm PEG Carboxyl (pentaerythritol
core), 8arm PEG Amine (hexaglycerin core), 8arm PEG Carboxyl
(hexaglycerin core), 8arm PEG Amine (tripentaerythritol core) and
8arm PEG Carboxyl (tripentaerythritol core). Preferred molecular
weights for such multi-arm PEG-derivatives in a backbone moiety are
1 kDa to 20 kDa, more preferably 2.5 kDa to 15 kDa and even more
preferably 5 kDa to 10 kDa.
[0157] Such additional functional groups may be provided by
dendritic moieties. Preferably, each dendritic moiety has a
molecular weight in the range of from 0.4 kDa to 4 kDa, more
preferably 0.4 kDa to 2 kDa. Preferably, each dendritic moiety has
at least 3 branchings and at least 4 functional groups, and at most
63 branchings and 64 functional groups, preferred at least 7
branchings and at least 8 reactive functional groups and at most 31
branchings and 32 functional groups.
[0158] Examples for such dendritic moieties are lysine, dilysine,
trilysine, tetralysine, pentalysine, hexylysine, heptalysine,
octalysine, nonalysine, decalysine, undecalysine, dodecalysine,
tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,
heptadecalysine, octadecalysine, nonadecalysine.
[0159] Also such multi-arm PEG derivatives may be connected to
dendritic moieties. Preferably, each dendritic moiety has a
molecular weight in the range of from 0.4 kDa to 2 kDa. Examples
for such dendritic moieties are lysine, dilysine, trilysine,
tetralysine, pentalysine, hexylysine, heptalysine, most preferred
trilysine, pentalysine or heptalysine.
[0160] Most preferably, the hydrogel carrier of the present
invention is characterized in that the backbone moiety has a
quarternary carbon of formula C(A-Hyp)4, wherein each A is
independently a poly(ethylene glycol) based polymeric chain
terminally attached to the quarternary carbon by a permanent
covalent bond and the distal end of the PEG-based polymeric chain
is covalently bound to a dendritic moiety Hyp, each dendritic
moiety Hyp having at least four functional groups representing the
interconnected functional groups and reactive functional
groups.
[0161] Preferably, each A is independently selected from the
formula --(CH2)n1(OCH2CH2)nX--, wherein n1 is 1 or 2; n is an
integer in the range of from 5 to 50; and X is a chemical
functional group covalently linking A and Hyp.
[0162] Preferably, A and Hyp are covalently linked by an amide
linkage.
[0163] Preferably, the dendritic moiety Hyp is a hyperbranched
polypeptide. Preferably, the hyperbranched polypeptide comprises
lysine in bound form. Preferably, each dendritic moiety Hyp has a
molecular weight in the range of from 0.4 kDa to 4 kDa. It is
understood that a backbone moiety C(A-Hyp)4 can consist of the same
or different dendritic moieties Hyp and that each Hyp can be chosen
independently. Each moiety Hyp consists of between 5 and 32
lysines, preferably of at least 7 lysines, i.e. each moiety Hyp is
comprised of between 5 and 32 lysines in bound form, preferably of
at least 7 lysines in bound form. Most preferably Hyp is comprised
of heptalysinyl.
[0164] The reaction of polymerizable functional groups a backbone
reagent, more specifically of Hyp with the polymerizable functional
groups of polyethyleneglycol based crosslinker reagents results in
a permanent amide bond.
[0165] Preferably, C(A-Hyp)4 has a molecular weight in the range of
from 1 kDa to 20 kDa, more preferably 1 kDa to 15 kDa and even more
preferably 1 kDa to 10 kDa.
[0166] One preferred backbone moiety is shown below, dashed lines
indicate interconnecting biodegradable linkages to crosslinker
moieties and n is an integer of from 5 to 50: backbone moiety has a
quarternary carbon of formula C(A-Hyp)4, wherein each A is
independently a polyethyleneglycol based polymeric chain terminally
attached to the quarternary carbon by a permanent covalent bond and
the distal end of the polymeric chain is covalently bound to a
dendritic moiety Hyp, each dendritic moiety Hyp having at least
four linkages representing the interconnected and biodegradable and
permanent linkages. Each backbone moiety contains at least 16
interconnected and biodegradable and permanent linkages, preferably
20 to 64 and more preferably 28 to 64 linkages.
[0167] Preferably, each A is independently selected from the
formula --(CH2)n1(OCH2CH2)nX--, wherein n1 is 1 or 2; n is an
integer in the range of from 5 to 50; and X is a functional group
covalently linking A and Hyp.
[0168] Preferably, A and Hyp are covalently linked by an amide
functional group.
[0169] Preferably, the dendritic moiety Hyp is a hyperbranched
polypeptide. Preferably, the hyperbranched polypeptide comprises
lysine, most preferably Hyp is undecalysinyl. Preferably, each
dendritic moiety Hyp has a molecular weight in the range of from
0.4 kDa to 4 kDa. It is understood that a backbone moiety C(A-Hyp)4
can consist of the same or different dendritic moieties Hyp and
that each Hyp can be chosen independently. Each moiety Hyp consists
of between 5 and 21 lysines, preferably of at least 7 lysines.
[0170] Also preferably, the hyperbranched polypeptide comprises
lysine, most preferably Hyp is heptalysinyl. Preferably, each
dendritic moiety Hyp has a molecular weight in the range of from
0.4 kDa to 2 kDa.
[0171] The sum of interconnecting biodegradable linkages and
permanent linkages connecting backbone moieties to prodrug-linker
and blocking groups can be equally or unequally divided by the
number of chains extending from the branching core.
[0172] Preferably, C(A-Hyp)4 has a molecular weight in the range of
from 1 kDa to 20 kDa, more preferably 1 kDa to 15 kDa and even more
preferably 1 kDa to 10 kDa.
[0173] Preferably, L2 is attached to Z through a thiosuccinimide
group or amide group, preferably an amide group, which in turn is
attached to the hydrogel's backbone moiety through a spacer, such
as an oligoethylene glycol chain. Preferably, the linkage of this
spacer chain to the backbone moiety is a permanent bond, preferably
an amide bond.
[0174] Biodegradability of the hydrogels according to the present
invention is achieved by introduction of hydrolytically degradable
bonds.
[0175] The terms "hydrolytically degradable", "biodegradable" or
"hydrolytically cleavable", "auto-cleavable", or "self-cleavage",
"self-cleavable", "transient" or "temporary" refers within the
context of the present invention to bonds and linkages which are
non-enzymatically hydrolytically degradable or cleavable under
physiological conditions (aqueous buffer at pH 7.4, 37.degree. C.)
with half-lives ranging from one hour to three months, including,
but are not limited to, aconityls, acetals, amides, carboxylic
anhydrides, esters, imines, hydrazones, maleamic acid amides, ortho
esters, phosphamides, phosphoesters, phosphosilyl esters, silyl
esters, sulfonic esters, aromatic carbamates, combinations thereof,
and the like.
[0176] If present in a hydrogel according to the invention as
degradable interconnected functional group, preferred biodegradable
linkages are esters, carbonates, phosphoesters and sulfonic acid
esters and most preferred are esters or carbonates.
[0177] The term "hydrolytically degradable" refers within the
context of the present invention to linkages which are
non-enzymatically hydrolytically degradable under physiological
conditions (aqueous buffer at pH 7.4, 37.degree. C.) with
half-lives ranging from one hour to three months, include, but are
not limited to, aconityls, acetals, carboxylic anhydrides, esters,
imines, hydrazones, maleamic acid amides, ortho esters,
phosphamides, phosphoesters, phosphosilyl esters, silyl esters,
sulfonic esters, aromatic carbamates, combinations thereof, and the
like. Preferred biodegradable linkages are esters, carbonates,
phosphoesters and sulfonic acid esters and most preferred are
esters or carbonates. It is understood that for in vitro studies
accelerated conditions like, for example, pH 9, 37.degree. C.,
aqueous buffer, may be used for practical purposes.
[0178] Permanent linkages are non-enzymatically hydrolytically
degradable under physiological conditions (aqueous buffer at pH
7.4, 37.degree. C.) with half-lives of six months or longer, such
as, for example, amides.
[0179] The degradation of the hydrogel is a multi-step reaction
where a multitude of degradable bonds is cleaved resulting in
degradation products which may be water-soluble or water-insoluble.
However each water-insoluble degradation product further comprises
degradable bonds so that it can be cleaved in that water-soluble
degradation products are obtained. These water-soluble degradation
products may comprise one or more backbone moieties. It is
understood that released backbone moieties may, for instance, be
permanently linked to spacer or blocking groups and/or
prodrug-linker degradation products.
[0180] The total amount of backbone moieties can be measured in
solution after complete degradation of the hydrogel, and during
degradation, fractions of soluble backbone degradation products can
be separated from the insoluble hydrogel and can be quantified
without interference from other soluble degradation products
released from the hydrogel. A hydrogel object may be separated from
excess water of buffer of physiological osmolality by sedimentation
or centrifugation. Centrifugation may be performed in such way that
the supernatant provides for at least 10% of the volume of the
swollen hydrogel. Soluble hydrogel degradation products remain in
the aqueous supernatant after such sedimentation or centrifugation
step, and water-soluble degradation products comprising one or more
backbone moieties are detectable by subjecting aliquots of such
supernatant to suitable separation and/or analytical methods. For
instance the backbone moieties may carry groups that exhibit UV
absorption at wavelengths where other degradation products do not
exhibit UV absorption. Such selectively UV-absorbing groups may be
structural components of the backbone moiety such as amide bonds or
may be introduced into the backbone by attachment to its reactive
functional groups by means of aromatic ring systems such as indoyl
groups.
[0181] In such hydrogel-linked prodrugs according to the invention,
it is desirable that almost all drug release (>90%) has occurred
before a significant amount of release of the backbone degradation
products (<10%) has taken place. This can be achieved by
adjusting the hydrogel-linked prodrug's half-life versus the
hydrogel degradation kinetics.
[0182] To introduce the hydrolytically cleavable bonds into the
hydrogel carrier of the invention, the backbone moieties can be
directly linked to each other by means of biodegradable bonds.
[0183] In one embodiment, the backbone moieties of the
biodegradable hydrogel carrier may be linked together directly,
i.e. without crosslinker moieties. The hyperbranched dendritic
moieties of two backbone moieties of such biodegradable hydrogel
may either be directly linked through an interconnected functional
group that connects the two hyperbranched dendritic moieties.
Alternatively, two hyperbranched dendritic moieties of two
different backbone moieties may be interconnected through two
spacer moieties connected to a backbone moiety and on the other
side connected to a crosslinking moiety separated by an
interconnected functional groups.
[0184] Alternatively, backbone moieties may be linked together
through crosslinker moieties, each crosslinker moiety is terminated
by at least two of the hydrolytically degradable bonds. In addition
to the terminating degradable bonds, the crosslinker moieties may
contain further biodegradable bonds. Thus, each end of the
crosslinker moiety linked to a backbone moiety comprises a
hydrolytically degradable bond, and additional biodegradable bonds
may optionally be present in the crosslinker moiety.
[0185] Preferably, the biodegradable hydrogel carrier is composed
of backbone moieties interconnected by hydrolytically degradable
bonds and the backbone moieties are linked together through
crosslinker moieties.
[0186] The biodegradable hydrogel carrier may contain one or more
different types of crosslinker moieties, preferably one. The
crosslinker moiety may be a linear or branched molecule and
preferably is a linear molecule. In a preferred embodiment of the
invention, the crosslinker moiety is connected to backbone moieties
by at least two biodegradable bonds.
[0187] If present in a hydrogel according to the invention as
degradable interconnected functional group, preferred biodegradable
linkages are carboxylic esters, carbonates, phosphoesters and
sulfonic acid esters and most preferred are carboxylic esters or
carbonates.
[0188] Preferably, crosslinker moieties have a molecular weight in
the range of from 60 Da to 5 kDa, more preferably, from 0.5 kDa to
4 kDa, even more preferably from 1 kDa to 4 kDa, even more
preferably from 1 kDa to 3 kDa. In one embodiment, a crosslinker
moiety consists of a polymer.
[0189] In addition to oligomeric or polymeric crosslinking
moieties, low-molecular weight crosslinking moieties may be used,
especially when hydrophilic high-molecular weight backbone moieties
are used for the formation of a biodegradable hydrogel according to
the invention.
[0190] Preferably, the poly(ethylene glycol) based crosslinker
moieties are hydrocarbon chains comprising ethylene glycol units,
optionally comprising further chemical functional groups, wherein
the poly(ethylene glycol) based crosslinker moieties comprise at
least each methylene glycol units, wherein m is an integer in the
range of from 3 to 100, preferably from 10 to 70. Preferably, the
poly(ethylene glycol) based crosslinker moieties have a molecular
weight in the range of from 0.5 kDa to 5 kDa.
[0191] If used in reference to a crosslinker moiety or a PEG-based
polymeric chain connected to a branching core, the term "PEG-based"
refers to a crosslinker moiety or PEG-based polymeric chain
comprising at least 20 weight % ethylene glycol moieties.
[0192] In one embodiment, monomers constituting the polymeric
crosslinker moieties are connected by biodegradable bonds. Such
polymeric crosslinker moieties may contain up to 100 biodegradable
bonds or more, depending on the molecular weight of the crosslinker
moiety and the molecular weight of the monomer units. Examples for
such crosslinker moieties are poly(lactic acid) or poly(glycolic
acid) based polymers. It is understood that such poly(lactic acid)
or poly(glycolic acid) chain may be terminated or interrupted by
alkyl or aryl groups and that they may optionally be substituted
with heteroatoms and chemical functional groups.
[0193] Preferably, the crosslinker moieties are PEG based,
preferably represented by only one PEG based molecular chain.
Preferably, the poly(ethylene glycol) based crosslinker moieties
are hydrocarbon chains comprising ethylene glycol units, optionally
comprising further chemical functional groups, wherein the
poly(ethylene glycol) based crosslinker moieties comprise at least
each methylene glycol units, wherein m is an integer in the range
of from 3 to 100, preferably from 10 to 70. Preferably, the
poly(ethylene glycol) based crosslinker moieties have a molecular
weight in the range of from 0.5 kDa to 5 kDa.
[0194] In a preferred embodiment of the present invention the
crosslinker moiety consists of PEG, which is symmetrically
connected through ester bonds to two alpha, omega-aliphatic
dicarboxylic spacers provided by backbone moieties connected to the
hyperbranched dendritic moiety through permanent amide bonds.
[0195] The dicarboxylic acids of the spacer moieties connected to a
backbone moiety and on the other side is connected to a
crosslinking moiety consist of 3 to 12 carbon atoms, most
preferably between 5 and 8 carbon atoms and may be substituted at
one or more carbon atom. Preferred substituents are alkyl groups,
hydroxyl groups or amido groups or substituted amino groups. One or
more of the aliphatic dicarboxylic acid's methylene groups may
optionally be substituted by O or NH or alkyl-substituted N.
Preferred alkyl is linear or branched alkyl with 1 to 6 carbon
atoms.
[0196] Preferably, there is a permanent amide bond between the
hyperbranched dendritic moiety and the spacer moiety connected to a
backbone moiety and on the other side is connected to a
crosslinking moiety.
[0197] One preferred crosslinker moiety is shown below; dashed
lines indicate interconnecting biodegradable linkages to backbone
moieties: [0198] , wherein n is an integer of from 5 to 50.
[0199] Preferably, the hydrogel carrier is composed of backbone
moieties interconnected by hydrolytically degradable bonds.
[0200] More preferably, the backbone moieties comprise a branching
core of the following formula: [0201] , [0202] wherein the dashed
line indicates attachment to the remainder of the backbone
moiety.
[0203] More preferably, the backbone moieties comprise a structure
of the following formula: [0204] , [0205] wherein n is an integer
of from 5 to 50 and the dashed line indicates attachment to the
remainder of the backbone moiety.
[0206] Preferably, backbone moiety comprises a hyperbranched moiety
Hyp.
[0207] More preferably, the backbone moiety comprises a
hyperbranched moiety Hyp of the following formula: [0208] , [0209]
wherein the dashed lines indicate attachment to the rest of the
molecule and carbon atoms marked with asterisks indicate
S-configuration.
[0210] Preferably, the backbone moieties are attached to at least
one spacer of the following formula: [0211] , [0212] wherein one of
the dashed lines indicates attachment to the hyperbranched moiety
Hyp and the second dashed line indicates attachment to the rest of
the molecule; and [0213] wherein m is an integer of from 2 to
4.
[0214] Preferably, the backbone moieties are linked together
through crosslinker moieties having the following structure
wherein q is an integer from 3 to 100;
[0215] Preferably, backbone moieties may be linked together through
crosslinker moieties, each crosslinker moiety being terminated by
at least two of the hydrolytically degradable bonds. In addition to
the terminating degradable bonds, the crosslinker moieties may
contain further biodegradable bonds. Thus, each end of the
crosslinker moiety linked to a backbone moiety shows a
hydrolytically degradable bond, and additional biodegradable bonds
may optionally be present in the crosslinker moiety.
[0216] The hydrogel may contain one or more different types of
crosslinker moieties, preferably one. The crosslinker moiety may be
a linear or branched molecule and preferably is a linear molecule.
In a preferred embodiment of the invention, the crosslinker moiety
is connected to backbone moieties by at least two biodegradable
bonds. The term biodegradable bond describes linkages that are
non-enzymatically hydrolytically degradable under physiological
conditions (aqueous buffer at pH 7.4, 37.degree. C.) with
half-lives ranging from one hour to three months, include, but are
not limited to, aconityls, acetals, carboxylic anhydrides, esters,
imines, hydrazones, maleamic acid amides, ortho esters,
phosphamides, phosphoesters, phosphosilyl esters, silyl esters,
sulfonic esters, aromatic carbamates, combinations thereof, and the
like. Preferred biodegradable linkages are esters, carbonates,
phosphoesters and sulfonic acid esters and most preferred are
esters or carbonates.
[0217] In one embodiment, a crosslinker moiety consists of a
polymer. Preferably, crosslinker moieties have a molecular weight
in the range of from 60 Da to 5 kDa, more preferably, from 60 Da to
4 kDa, even more preferably from 60 Da to 3 kDa.
[0218] Also preferably, crosslinker moieties have a molecular
weight in the range of from 0.5 kDa to 5 kDa, more preferably, from
1 kDa to 4 kDa, even more preferably from 1 kDa to 3 kDa.
[0219] In addition to oligomeric or polymeric crosslinking
moieties, low-molecular weight crosslinking moieties may be used,
especially when hydrophilic high-molecular weight backbone moieties
are used for the hydrogel formation.
[0220] Preferably, the polyethyleneglycol based crosslinker
moieties are hydrocarbon chains comprising ethylene glycol units,
optionally comprising further functional groups, wherein the
polyethyleneglycol based crosslinker moieties comprise at least
each methylene glycol units, wherein m is an integer in the range
of from 1 to 70. Preferably, the polyethyleneglycol based
crosslinker moieties have a molecular weight in the range of from
60 Da to 5 kDa. Also preferably, m is an integer in the range of
from 10 to 70. Preferably, the polyethyleneglycol based crosslinker
moieties have a molecular weight in the range of from 0.5 kDa to 5
kDa.
[0221] Preferably, the crosslinker moieties are PEG-based,
preferably represented by only one PEG-based molecular chain.
Preferably, the polyethyleneglycol-based crosslinkers are
hydrocarbon chains comprising one or more ethylene glycol units,
optionally comprising further functional groups, wherein the
polyethyleneglycol based crosslinker moieties comprise at least
each methylene glycol units, wherein m is an integer in the range
of from 1 to 70. Preferably, the polyethyleneglycol based
crosslinkers have a molecular weight in the range of from 60 Da to
5 kDa.
[0222] In one embodiment, monomers constituting the polymeric
crosslinker moieties are connected by biodegradable bonds. Such
polymeric crosslinkers may contain up to 100 biodegradable bonds or
more, depending on the molecular weight of the crosslinker moiety
and the molecular weight of the monomer units. Examples for such
crosslinkers are polylactic acid or polyglycolic acid based.
[0223] Also preferably, the crosslinker moieties are PEG based,
preferably represented by only one PEG based molecular chain.
Preferably, the polyethyleneglycol based crosslinkers are
hydrocarbon chains comprising ethylene glycol units, optionally
comprising further functional groups, wherein the
polyethyleneglycol based crosslinker moieties comprise at least
each methylene glycol units, wherein m is an integer in the range
of from 10 to 70. Preferably, the polyethyleneglycol based
crosslinkers have a molecular weight in the range of from 0.5 kDa
to 5 kDa.
[0224] In a preferred embodiment of the present invention the
crosslinker moiety consists of a PEG chain, which is symmetrically
connected through ester bonds to two alpha, omega-aliphatic
dicarboxylic spacers provided by backbone moieties through
permanent amide bonds. The dicarboxylic acids consists of 3 to 12
carbon atoms, most preferably between 5 and 8 carbon atoms and may
be substituted at one or more carbon atom. Preferred substituents
are alkyl groups, hydroxy groups or amido groups or substituted
amino groups. One or more of the aliphatic dicarboxylic acid's
methylene groups may optionally be substituted by O or NH or
alkyl-substituted N. Preferred alkyl is linear or branched alkyl
with 1 to 6 carbon atoms.
[0225] the hydrolysis rate of the biodegradable bonds between
backbone moieties and crosslinker moieties is influenced or
determined by the number and type of connected atoms adjacent to
the PEG-ester carboxy group. For instance, by selecting from
succinic, adipic or glutaric acid for PEG ester formation it is
possible to vary the degradation half-lives of the biodegradable
hydrogel carrier according to the invention.
[0226] The degradation of the biodegradable hydrogel carrier
according to the invention is a multi-step reaction where a
multitude of degradable bonds is cleaved resulting in degradation
products which may be water-soluble or water-insoluble. However,
water-insoluble degradation products may further comprise
degradable bonds so that they can be cleaved in that water-soluble
degradation products are obtained. These water-soluble degradation
products may comprise one or more backbone moieties. It is
understood that released backbone moieties may, for instance, be
permanently conjugated to spacer or blocking or linker groups or
affinity groups and/or prodrug linker degradation products and that
also water-soluble degradation products may comprise degradable
bonds.
[0227] The structures of the branching core, PEG-based polymeric
chains, hyperbranched dendritic moieties and moieties attached to
the hyperbranched dendritic moieties can be inferred from the
corresponding descriptions provided in the sections covering the
hydrogel carriers of the present invention. It is understood that
the structure of a degradant depends on the type of hydrogel
according to the invention undergoing degradation.
[0228] The total amount of backbone moieties can be measured in
solution after complete degradation of the hydrogel according to
the invention, and during degradation, fractions of soluble
backbone degradation products can be separated from the insoluble
hydrogel according to the invention and can be quantified without
interference from other soluble degradation products released from
the hydrogel according to the invention. A hydrogel object
according to the invention may be separated from excess water of
buffer of physiological osmolality by sedimentation or
centrifugation. Centrifugation may be performed in such way that
the supernatant provides for at least 10% of the volume of the
swollen hydrogel according to the invention. Soluble hydrogel
degradation products remain in the aqueous supernatant after such
sedimentation or centrifugation step, and water-soluble degradation
products comprising one or more backbone moieties are detectable by
subjecting aliquots of such supernatant to suitable separation
and/or analytical methods.
[0229] Preferably, water-soluble degradation products may be
separated from water-insoluble degradation products by filtration
through 0.45 .mu.m filters, after which the water-soluble
degradation products can be found in the flow-through.
Water-soluble degradation products may also be separated from
water-insoluble degradation products by a combination of a
centrifugation and a filtration step.
[0230] For instance the backbone moieties may carry groups that
exhibit UV absorption at wavelengths where other degradation
products do not exhibit UV absorption. Such selectively
UV-absorbing groups may be structural components of the backbone
moiety such as amide bonds or may be introduced into the backbone
by attachment to its reactive functional groups by means of
aromatic ring systems such as indoyl groups.
[0231] In such hydrogel-linked prodrugs according to the invention,
it is desirable that almost all drug release (>90%) has occurred
before a significant amount of release of the backbone degradation
products (<10%) has taken place. This can be achieved by
adjusting the hydrogel-linked prodrug's half-life versus the
hydrogel degradation kinetics.
[0232] The hydrogel-linked prodrug of the present invention can be
prepared starting from the hydrogel of the present invention by
convenient methods known in the art. It is clear to a practitioner
in the art that several routes exist. For example the prodrug
linker mentioned above to which the biologically active moiety is
covalently attached can be reacted with the reactive functional
groups of the hydrogel of the present invention with or with
already bearing the active moiety in part or as whole.
[0233] In a preferable method of preparation, the hydrogel is
generated through chemical ligation reactions. The hydrogel may be
formed from two macromolecular educts with complementary
functionalities which undergo a reaction such as a condensation or
addition. One of these starting materials is a crosslinker reagent
with at least two identical functional groups and the other
starting material is a homomultifunctional backbone reagent.
Suitable functional groups present on the crosslinker reagent
include terminal amino, carboxylic acid and derivatives, maleimide
and other alpha,beta unsaturated Michael acceptors like
vinylsulfone, thiol, hydroxyl groups. Suitable functional groups
present in the backbone reagent include but are not limited to
amino, carboxylic acid and derivatives, maleimide and other
alpha,beta unsaturated Michael acceptors like vinylsulfone, thiol,
hydroxyl groups.
[0234] If the crosslinker reagent reactive functional groups are
used substoichiometrically with respect to backbone reactive
functional groups, the resulting hydrogel will be a reactive
hydrogel with free reactive functional groups attached to the
backbone structure.
[0235] Optionally, the linker may be first conjugated to the drug
compound and the resulting prodrug linker conjugate may then react
with the hydrogel's reactive functional groups. Alternatively,
after activation of one of the functional groups of the linker, the
linker-hydrogel conjugate may be contacted with drug compound in
the second reaction step and excess drug may be removed by
filtration after conjugation of the drug to the hydrogel-bound
linker.
[0236] A preferred process for the preparation of a prodrug
according to the present invention is as follows:
[0237] A preferred starting material for the backbone reagent
synthesis is a 4-arm PEG tetra amine or 8-arm PEG octa amine, with
the PEG reagent having a molecular weight ranging from 2000 to
10000 Dalton, most preferably fom 2000 to 5000 Da. To such
multi-arm PEG-derivatives, lysine residues are coupled sequentially
to form the hyperbranched backbone reagent. It is understood that
the lysines can be partially or fully protected by protective
groups during the coupling steps and that also the final backbone
reagent may contain protective groups. A preferred building block
is bis-boc lysine. Alternatively, instead of sequential additions
of lysine residues, a dendritic poly-lysine moiety may be assembled
first and subsequently coupled to the 4-arm PEG tetra amine or
8-arm PEG octa amine. It is desirable to obtain backbone reagent
carrying 32 amino groups, consequently seven lysines would be
attached to each arm of a 4-arm PEG, or five lysines would be
attached to each arm of a 8-arm PEG. In another embodiment, the
multi-arm PEG derivative is a tetra- or octa carboxy PEG. In this
case, the dendritic moieties may be generated from glutaric or
aspartic acid, and the resulting backbone reagent would carry 32
carboxy groups. It is understood that all or a fraction of the
backbone reagent's functional groups may be present in a free form,
as salts or conjugated to protecting groups. It is understood that
due to practical reasons the backbone reagent's number of lysines
per PEG-arm will be between six and seven, more preferably
approximately seven.
[0238] A preferred backbone reagent is shown below:
##STR00012##
[0239] Synthesis of the crosslinker reagent starts from a linear
PEG chain with a molecular weight ranging from 0.2 to 5 kDa, more
preferably from 0.6 to 2 kDa, which is esterified with a half ester
of a dicarboxylic acid, most adipic acid or glutaric acid.
Preferred protecting group for half ester formation is the benzylic
group. The resulting bis dicarboxylic acid PEG half esters are
converted into more reactive carboxy compounds such as acyl
chlorides or active esters, eg pentafluorophenyl or
N-hydroxysuccinimide esters, most preferred N-hydroxysuccinimde
esters, of which preferred selected structure is shown below.
##STR00013##
wherein each m independently is an integer ranging from 2 to 4, and
q is an integer of from 3 to 100.
[0240] More preferred is the following structure:
##STR00014##
[0241] Alternatively, the bis dicarboxylic acid PEG half esters may
be activated in the presence of a coupling agent such as DCC or
HOBt or PyBOP.
[0242] In an alternative embodiment the backbone reagent carries
carboxyl groups and the corresponding crosslinker reagent would be
selected from ester-containing amino-terminated PEG-chains.
[0243] Backbone reagent and crosslinker reagent may be polymerized
to form the hydrogel according to the invention using inverse
emulsion polymerization. After selecting the desired stoichiometry
between backbone and crosslinker polymerizable groups, backbone and
crosslinker are dissolved in DMSO and a suitable emulgator with an
appropriately selected HLB value, preferably Arlacel P135, is
employed to form an inverse emulsion using a mechanical stirrer and
controlling the stirring speed. Polymerization is initiated by the
addition of a suitable base, preferably by
N,N,N',N'-tetramethylethylenene diamine. After stirring for an
appropriate amount of time, the reaction is quenched by the
addition of an acid, such as acetic acid and water. The beads are
harvested, washed, and fractionated according to particle size by
mechanical sieving. Optionally, protecting groups may be removed at
this stage.
[0244] In an alternative embodiment of this invention,
multi-functional moieties are coupled to the reactive functional
groups of the polymerized reactive hydrogel to increase the number
of functional groups which allows to increase the drug load of the
hydrogel. Such multi-functional moieties may be provided by
suitably substituted derivatives of lysine, dilysine, trilysine,
tetralysine, pentalysine, hexylysine, heptalysine, or oligolysine,
low-molecular weight PEI. Preferably, the multi-functional moiety
is lysine.
[0245] Further, such hydrogel according to the invention may be
functionalized with a spacer carrying the same functional group,
for instance, amino groups may be introduced into the hydrogel by
coupling a heterobifunctional spacer, such as suitably activated
COOH-(EG).sub.6-NH-fmoc (EG=ethylene glycol), and removing the
fmoc-protecting group.
[0246] In yet another embodiment, a drug compound is first
conjugated to a linker in such a fashion that the linkage between
drug compound and linker is a covalent transient linkage such as an
aromatic amide linkage, and is subsequently reacted with a reactive
biodegradable hydrogel to form a prodrug according to the
invention.
[0247] Further, such hydrogel according to the invention may be
functionalized with a spacer carrying a different reactive
functional group than provided by the hydrogel. For instance,
maleimide reactive functional groups may be introduced into the
hydrogel by coupling a suitable heterobifunctional spacer such as
Mal-(EG).sub.6-NHS to the hydrogel. Such functionalized hydrogel
can be further conjugated to drug-linker reagents, carrying a
reactive thiol group on the linker moiety to form carrier-linked
prodrugs according to the present invention.
[0248] After loading the drug-linker conjugate to the
functionalized maleimido group-containing hydrogel, all remaining
functional groups are capped with a suitable blocking reagents,
such as mercaptoethanol, to prevent undesired side-reactions.
[0249] A particularly preferred method for the preparation of a
prodrug of the present invention comprises the steps of
(a) reacting a compound of formula C(A'-X.sup.1).sub.4, wherein
A'-X.sup.1 represents A before its binding to Hyp or a precursor of
Hyp and X.sup.1 is a suitable chemical functional group, with a
compound of formula Hyp'-X.sup.2, wherein Hyp'-X.sup.2 represents
Hyp before its binding to A or a precursor of Hyp and X.sup.2 is a
suitable chemical functional group to react with X.sup.1; (b)
optionally reacting the resulting compound from step (a) in one or
more further steps to yield a compound of formula C(A-Hyp).sub.4
having at least four chemical functional groups; (c) reacting the
at least four chemical functional groups of the resulting compound
from step (b) with a poly(ethylene glycol) based crosslinker
precursor reagent, wherein the crosslinker precursor reagent is
used in a sub-stoichiometric amount compared to the total number of
functional groups of C(A-Hyp).sub.4 to yield a hydrogel according
to the invention; (d) reacting remaining un-reacted reactive
functional groups (representing the reactive functional groups of
the backbone comprised in the reactive biodegradable hydrogel of
the present invention) in the hydrogel backbone of step (c) with a
covalent conjugate of biologically active moiety and transient
prodrug linker or first reacting the un-reacted reactive functional
groups with the transient prodrug linker and subsequently with the
biologically active moiety; (e) optionally capping remaining
un-reacted reactive functional groups to yield a prodrug of the
present invention.
[0250] Specifically, hydrogels of the present invention are
synthesized as follows:
[0251] For bulk polymerization, backbone reagent and crosslinker
reagent are mixed in a ratio amine/active ester of 2:1 to
1.05:1.
[0252] Both backbone reagent and crosslinker reagent are dissolved
in DMSO to give a solution with a concentration of 5 to 50 g per
100 mL, preferably 7.5 to 20 g per 100 ml and most preferably 10 to
20 g per 100 ml.
[0253] To effect polymerization, 2 to 10% (vol.)
N,N,N',N'-tertramethylethylene diamine (TMEDA) are added to the
DMSO solution containing crosslinker reagent and backbone reagent
and the mixture is shaken for 1 to 20 sec and left standing. The
mixture solidifies within less than 1 min.
[0254] Such hydrogel according to the invention is preferably
comminuted by mechanical processes such as stirring, crushing,
cutting pressing, or milling, and optionally sieving.
[0255] For emulsion polymerization, the reaction mixture is
comprised of the dispersed phase and the continuous phase.
[0256] For the dispersed phase, backbone reagent and crosslinker
reagent are mixed in a ratio amine/active ester of 2:1 to 1.05:1
and are dissolved in DMSO to give a to give a solution with a
concentration of 5 to 50 g per 100 mL, preferably 7.5 to 20 g per
100 ml and most preferably 10 to 20 g per 100 ml.
[0257] The continuous phase is any solvent, that is not miscible
with DMSO, not basic, aprotic and shows a viscosity lower than 10
Pa*s. Preferably, the solvent is not miscible with DMSO, not basic,
aprotic, shows a viscosity lower than 2 Pa*s and is non-toxic. More
preferably, the solvent is a saturated linear or branched
hydrocarbon with 5 to 10 carbon atoms. Most preferably, the solvent
is n-heptane.
[0258] To form an emulsion of the dispersed phase in the continuous
phase, an emulsifier is added to the continuous phase before adding
the dispersed phase. The amount of emulsifier is 2 to 50 mg per mL
dispersed phase, more preferably 5 to 20 mg per mL dispersed phase,
most preferably 10 mg per mL dispersed phase.
[0259] The emulsifier has an HLB-value of 3 to 8. Preferably, the
emulsifier is a triester of sorbitol and a fatty acid or an
poly(hydroxyl fatty acid)-poly(ethylene glycol) conjugate. More
preferably, the emulsifier is an poly(hydroxy-fatty
acid)-polyethylene glycol conjugate, with a linear poly(ethylene
glycol) of a molecular weight in the range of from 0.5 kDa to 5 kDa
and poly(hydroxy-fatty acid) units of a molecular weight in the
range of from 0.5 kDa to 3 kDa on each end of the chain. Most
preferably, the emulsifier is poly(ethylene glycol) dipolyhydroxy
stearate, Cithrol DPHS (Cithrol DPHS, former Arlacel P135, Croda
International Plc).
[0260] Droplets of the dispersed phase are generated by stirring
with an axial flow impeller with a geometry similar to stirrers
such as Isojet, Intermig, Propeller (EKATO Ruhr- and Mischtechnik
GmbH, Germany)), most preferably similar to Isojet with a diameter
of 50 to 90% of the reactor diameter. Preferably, stirring is
initated before addition of the dispersed phase. Stirrer speed is
set to 0.6 to 1.7 m/s. The dispersed phase is added at room
temperature, and the concentration of the disperse phase is 2% to
70%, preferably 5 to 50%, more preferably 10 to 40%, and most
preferably 20 to 35% of the total reaction volume. The mixture of
dispersed phase, emulsifier and continuous phase is stirred for 5
to 60 min before adding the base to the effect polymerization.
[0261] 5 to 10 equivalents (referred to each amide bond to be
formed) of a base are added to the mixture of dispersed and
continuous phase. The base is aprotic, non nucleophilic and soluble
in the disperse phase. Preferably, the base is aprotic, non
nucleophilic, well soluble in both disperse phase and DMSO. More
preferably, the base is aprotic, non nucleophilic, well soluble in
both disperse phase and DMSO, an amine base and non-toxic. Most
preferably, the base is N,N,N',N'-tertramethylethylene diamine
(TMEDA). Stirring in the presence of base is continued for 1 to 16
h.
[0262] During stirring, droplets of dispersed phase are hardened to
become crosslinked hydrogel beads according to the invention which
can be collected and fractionation according to size is performed
on a vibrational continuous sieving machine with a 75 .mu.m and a
32 .mu.m deck to give hydrogel microparticles according to the
invention.
[0263] The hydrogel for the prodrug of the present invention can be
obtained from the preparation methods in form of micro-particles.
In a preferred embodiment of the invention, the reactive hydrogel
is a shaped article such as a mesh or a stent. Most preferably, the
hydrogel is formed into microparticulate beads which can be
administered as subcutaneous or intramuscular injectably by means
of a standard syringe. Such soft beads may have a diameter of
between 1 and 500 micrometer.
[0264] Preferably, such beaded hydrogel prodrugs have a diameter of
between 10 and 100 micrometer if suspended in an isotonic aqueous
formulation buffer, most preferably a diameter of between 20 and
100 micrometer, most preferably a diameter of between 25 and 80
micrometer.
[0265] Preferably, such beaded biodegradable hydrogel prodrugs can
be administered by injection through a needle smaller than 0.6 mm
inner diameter, preferably through a needle smaller than 0.3 mm
inner diameter, more preferably through a needle small than 0.25 mm
inner diameter, even more preferably through a needle smaller than
0.2 mm inner diameter, and most preferably through a needle small
than 0.16 mm inner diameter.
[0266] It is understood that the terms "can be administered by
injection", "injectable" or "injectability" refer to a combination
of factors such as a certain force applied to a plunger of a
syringe containing the biodegradable hydrogel according to the
invention swollen in a liquid at a certain concentration (w/v) and
at a certain temperature, a needle of a given inner diameter
connected to the outlet of such syringe, and the time required to
extrude a certain volume of the biodegradable hydrogel carrier
according to the invention from the syringe through the needle.
[0267] In order to provide for injectability, a volume of 1 mL of
the prodrugs according to the invention swollen in water to a
concentration of at least 5% (w/v) and contained in a syringe
holding a plunger of a diameter of 4.7 mm can be extruded at room
temperature within 10 seconds by applying a force of less than 50
Newton.
[0268] Preferably injectability is achieved for a hydrogel prodrug
according to the invention swollen in water to a concentration of
ca. 10% (w/v).
[0269] Within one embodiment of the present invention, in case
X.sup.1 is a cyclic fragment and X.sup.2 is C(R.sup.3R.sup.3a), the
order of X.sup.1 (the X.sup.1 fragment) and X.sup.2 (the X.sup.2
fragment) within the moiety L.sup.1 may be changed. This means that
in such a case the moiety L.sup.1 is represented by formula
(Ia),
##STR00015##
wherein (besides X.sup.1 and X.sup.2) all substituents (such as
R.sup.2) and fragments (such as L.sup.2) have the same (chemical)
definitions as indicated within the context of the present
invention for formula (I).
[0270] The drug linker conjugate D-L is any combination of the
aromatic amine containing biologically active moiety D and the
non-biologically active linker (both) as defined above, wherein the
dashed line indicates the attachment of L.sup.1 to an aromatic
amino group of D by forming an amide bond.
[0271] Another subject of the present invention is a method for the
synthesis of a prodrug or a pharmaceutically acceptable salt
thereof as defined above. Prodrugs or precursors of prodrugs
according to the present invention may be prepared by known methods
or in accordance with the reaction sequences described below. The
starting materials used in the preparation (synthesis) of prodrugs
of the invention or precursors thereof are known or commercially
available, or can be prepared by known methods or as described
below.
[0272] All reactions for the synthesis of the prodrugs according to
the present invention including precursors such as the moiety
L.sup.1 according to the formula (I) are per se well-known to the
skilled person and can be carried out under standard conditions
according to or analogously to procedures described in the
literature, for example in Houben-Weyl, Methoden der Organischen
Chemie (Methods of Organic Chemistry), Thieme-Verlag, Stuttgart, or
Organic Reactions, John Wiley & Sons, New York. Depending on
the circumstances of the individual case, in order to avoid side
reactions during the synthesis of a prodrug or a precursor thereof,
it can be necessary or advantageous to temporarily block functional
groups by introducing protective groups and to deprotect them in a
later stage of the synthesis, or introduce functional groups in the
form of precursor groups which in a later reaction step are
converted into the desired functional groups. Such synthesis
strategies and protective groups and precursor groups which are
suitable in an individual case are known to the skilled person. If
desired, the prodrugs or precursors can be purified by customary
purification procedures, for example by recrystallization or
chromatography.
[0273] The prodrugs according to the present invention (or a
pharmaceutically acceptable salt thereof) may be prepared by a
method comprising the step of reacting a prodrug precursor L-Y with
a biologically active drug D-H to obtain the drug linker conjugate
D-L by forming an amide bond, wherein Y is a leaving group.
[0274] In respect of the prodrug precursor L-Y, L has the same
meaning as indicated above in connection with the drug linker
conjugate D-L. The same holds true for the analogous employment of
the prodrug precursor L.sup.1-Y in respect of the moiety L.sup.1
represented by formula (I) or formula (Ia), respectively.
[0275] Y is a leaving group. Such leaving groups are known to a
person skilled in the art. Preferably, Y is chloride, bromide,
fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl,
N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl,
pentafluorophenoxy, 2-thiooxo-thiazolidinyl, or
N-hydroxysulfosuccinimidyl.
[0276] In case the synthesis of a prodrug according to the present
invention is carried out by employing a precursor L.sup.1-Y, a drug
linker intermediate (L.sup.1-D) is obtained by reacting L.sup.1-Y
with the biologically active drug D-H (by forming an amide bond).
In such a case, said drug linker intermediate L.sup.1-D is reacted
further to obtain the drug linker conjugate D-L by adding the
moiety L.sup.2 and the carrier group Z to said drug linker
intermediate L.sup.1-D. It has to be indicated that the addition of
L.sup.2 and/or Z to L.sup.1-D may be performed in several steps by
preparing further intermediate compounds prior to obtaining the
drug linker conjugate D-L.
[0277] Alternatively, a prodrug precursor L*-Y may be employed
instead of L.sup.1-Y, wherein L* is selected from a fragment of
L.sup.1, L.sup.1 containing at least one protecting group or
L.sup.1 additionally containing precursors of L.sup.2 and/or Z.
[0278] Another subject of the present invention is the use of
prodrugs (or a pharmaceutically acceptable salt thereof) comprising
a drug linker conjugate D-L as pharmaceuticals or medicaments,
respectively. With respect of the definitions of the drug linker
conjugate D-L (as well as further substituents such as L.sup.1 or
X.sup.1) the same explanations as laid out above in the context of
the prodrug as such apply.
[0279] Another subject of the present invention is a pharmaceutical
composition comprising an effective dose of at least one prodrug
(or a pharmaceutically acceptable salt thereof) as defined above
and a pharmaceutically acceptable excipient. Furthermore, the
present invention also comprises the use of such pharmaceutical
compositions as pharmaceuticals or medicaments, respectively.
[0280] Examples of diseases, which can be treated by employing the
prodrugs and/or the pharmaceutical compositions according to the
present invention are dopamine receptor related diseases, including
Parkinson's disease, neurological disorders, amyotrophic lateral
sclerosis, compulsive behavior, bipolar disorders, Tourette's
syndrome, depressive disorders, treatment resistant depression,
fibromyalia or restless leg syndrome (RLS).
[0281] Preferred diseases to be treated are Parkinson's disease and
RLS.
[0282] The use of the prodrugs and/or the pharmaceutical
compositions according to the present invention includes the
prophylaxis and/or treatment of said diseases. The present
invention also includes a method for producing a medicament for the
prophylaxis and/or treatment of said diseases. The present
invention also includes a method of treating, controlling, delaying
or preventing in a mammalian patient in need of the treatment of
one or more conditions comprising administering to said patient a
therapeutically effective amount of a prodrug (or a
pharmaceutically acceptable salt thereof) according to the present
invention or a respective pharmaceutical composition.
[0283] All prodrugs according to the present invention or the
respective pharmaceutical compositions can be administered to
animals, preferably to mammals, and in particular to humans. The
prodrugs and/or pharmaceutical compositions can be administered as
such or in mixtures with one another or in mixtures with other
pharmaceuticals. The prodrugs and/or the respective pharmaceutical
compositions according to the present invention are administered in
effective doses, which are known to a person skilled in the
art.
[0284] The following examples illustrate the invention without
limitation.
EXAMPLES
Materials and Methods
[0285] 2-Chlorotrityl chloride resin and Sieber amide resin were
obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany.
Boc-Gly-OH and Fmoc-Gly-OH were obtained from Merck KGaA,
Darmstadt, Germany. Mal-dPEG.sub.6-NHS-ester was obtained from
celares GmbH, Berlin, Germany. Pramipexole dihydrochloride was
optained from Carbone Scientific Co., Ltd., Wuhan, China.
[0286] All other chemicals were obtained from Sigma-ALDRICH Chemie
GmbH, Taufkirchen, Germany.
[0287] Solid phase synthesis was performed on 2-Chlorotrityl
chloride resin with a loading of 1.1 mmol/g or Sieber amide resin
with a loading of 0.64 mmol/g. Syringes equipped with polypropylene
frits were used as reaction vessels.
[0288] Loading of the first amino acid to resins was performed
according to manufacturer's instructions.
Fmoc Deprotection:
[0289] For Fmoc protecting-group removal, the resin was agitated
with 2/2/96 (v/v/v) piperidine/DBU/DMF (two times, 10 min each) and
washed with DMF (ten times).
Standard Coupling Conditions for Acids:
[0290] Coupling of acids (aliphatic acids, Fmoc-amino acids) to
free amino groups on resin was achieved by agitating resin with 2
eq of acid, 2 eq PyBOP and 4 eq DIEA in relation to free amino
groups on resin (calculated based on theoretical loading of the
resin) in DMF at room temperature. After 1 hour resin was washed
with DMF (10 times).
3-Maleimido Propionic Acid Coupling:
[0291] Coupling of 3-maleimido propionic acid to free amino groups
on resin was achieved by agitating resin with 2 eq of acid, 2 eq
DIC and 2 eq HOBt in relation to free amino groups in DMF at room
temperature. After 30 min, the resin was washed with DMF (10
times).
Standard Protocol for the Synthesis of Ureas on Resin:
[0292] Synthesis of ureas on resin was achieved by agitating resin
with 2.5 eq of bis(pentafluorophenyl) carbonate and 5 eq DIEA in
relation to free amino groups in DCM at room temperature. After 45
min resin was washed with DMF (10 times). 1 eq of amine and 2.5 eq
DIEA were dissolved in DCM. Mixture was added to resin and agitated
for 75 min at room temperature. Resin was washed with DMF (10
times).
Cleavage Protocol for Sieber Amide Resin:
[0293] Upon completed synthesis, the resin was washed with DCM (10
times), dried in vacuo and treated repeatedly (three times a 15
minutes) with 96/2/2 (v/v) DCM/TES/TFA. Eluates were combined,
volatiles were removed under a nitrogen stream and product was
purified by RP-HPLC. HPLC fractions containing product were
combined and lyophilized.
Cleavage Protocol for 2-Chlorotrityl Chloride Resin:
[0294] Upon completed synthesis, the resin was washed with DCM,
dried in vacuo and treated three times for 30 minutes with 7/3
(v/v) DCM/HFIP. Eluates were combined, volatiles were removed under
a nitrogen stream and product was purified by RP-HPLC. HPLC
fractions containing product were combined and lyophilized.
RP-HPLC Purification:
[0295] RP-HPLC was done on a 100.times.20 or a 100.times.40 mm C18
ReproSil-Pur 3000DS-3 5.mu. column (Dr. Maisch, Ammerbuch, Germany)
connected to a Waters 600 HPLC System and Waters 2487 Absorbance
detector. Linear gradients of solution A (0.1% TFA in H.sub.2O) and
solution B (0.1% TFA in acetonitrile or 0.1% TFA in 2/1 (v/v)
methanol/isopropanol) were used. HPLC fractions containing product
were lyophilized.
[0296] Analytics: Ultra performance liquid
chromatography-electronspray ionization mass spectrometry
(UPLC-ESI-MS) was performed on a Waters Acquity Ultra Performance
LC instrument connected to a Thermo scientific LTQ Orbitrap
Discovery instrument and spectra were, if necessary, interpreted by
Thermo scientific software xcalibur.
[0297] Mass spectra of PEG products showed a series of
(CH.sub.2CH.sub.2O).sub.n moieties due to polydispersity of PEG
staring materials. For easier interpretation only one single
representative m/z signal is given in the examples.
Example 1
Synthesis of Linker Pramipexole Conjugate (1b)
Synthesis of Intermediate (1a)
##STR00016##
[0299] S-tritylcysteamine (100 mg, 0.313 mmol), succinic anhydride
(323 mg, 3.130 mmol) and DIEA (273 .mu.L, 1.567 mmol) were
dissolved in dry DCM (2.2 mL) and agitated for 30 min at room
temperature. The mixture was acidified by addition of AcOH (0.7
mL), diluted with diethyl ether and washed twice with water. The
organic phase was dried over MgSO.sub.4, the solvent was evaporated
under reduced pressure.
[0300] Yield: 95 mg (0.226 mmol).
[0301] MS: m/z 442.1=[M+Na].sup.+ (calculated=442.5 g/mol).
Synthesis of (1b)
##STR00017##
[0303] Reagent 1a (30 mg, 0.072 mmol), PyBOP (45 mg, 0.086 mmol)
and N-methyl morpholine (79 .mu.L, 0.715 mmol) were dissolved in
DMSO (1 mL). Pramipexole dihydrochloride (81 mg, 0.286 mmol) was
added and the mixture was stirred for 16 h. The reaction was
quenched by addition of acetic acid and the mixture was diluted
with 3.5 mL acetonirile/water 1/1+0.1% TFA. The trityl protected
intermediate of 1b was purified by RP-HPLC. After lyophilisation 21
mg (0.029 mmol) of the TFA salt were obtained.
[0304] MS: m/z 613.4=[M+H].sup.+ (calculated=613.9 g/mol).
[0305] For trityl deprotection the lyophilisate was dissolved in
HFIP (2 mL), TES (20 .mu.L) was added, and the mixture was
incubated for 10 min. Volatiles were evaporated and 1b was purified
by RP-HPLC.
[0306] Yield: 12 mg (0.025 mmol, TFA salt).
[0307] MS: m/z 371.2=[M+H].sup.+ (MW calculated=371.2 g/mol).
Example 2
Synthesis of Linker Pramipexole Conjugate (2b)
Synthesis of Intermediate (2a)
##STR00018##
[0309] 2a was synthesized as described for 1a except for the use of
6-(tritylthio)hexane-1-amine instead of trityl cysteamine.
[0310] Yield: 170 mg (0.226 mmol).
[0311] MS: m/z 498.2=[M+Na].sub.+, (calculated=498.6 g/mol).
Synthesis of (2b)
##STR00019##
[0313] 2b was synthesized as described for 1b except for the use of
2a instead of 1a.
[0314] 2b: Yield: 3.5 mg (0.006 mmol, TFA salt).
[0315] MS: m/z 427.2=[M+H].sup.+, (MW calculated=427.2 g/mol).
Example 3
Synthesis of Intermediate (3a)
##STR00020##
[0317] 3a was synthesized as described for 2a except for the use of
1,4-dioxane-2,6-dione instead of succinic anhydride.
[0318] Yield: 117 mg (0.237 mmol).
[0319] MS: m/z 983.4=[2M+H].sup.+ (calculated=984.2 g/mol).
Synthesis of Linker Pramipexole Intermediate (3b)
##STR00021##
[0321] 3b was synthesized as described for 1b except for the use of
3a instead of 1a. The coupling of pramipexole was completed within
30 min.
[0322] 3b: Yield: 4.5 mg (0.008 mmol, TFA salt).
[0323] MS: m/z 443.2=[M+H].sup.+, (calculated=443.7 g/mol).
Example 4
Synthesis of Linker Pramipexol Conjugate (4b)
Synthesis of Intermediate (4a)
##STR00022##
[0325] 4a was synthesized as described for 2a except for the use of
tert-butyl 2,6-dioxomorpholine-4-carboxylate instead of succinic
anhydride.
[0326] Yield: 148 mg (0.250 mmol).
[0327] MS: m/z 591.3=[M+H].sup.+, (calculated=591.8 g/mol).
Synthesis of (4b)
##STR00023##
[0329] 4b was synthesized as described for 1b except for the use of
4a instead of 1a. The coupling of pramipexole was completed within
40 min.
[0330] Prior trityl and boc deprotection 38 mg (0.042 mmol, TFA
salt) of the intermediate were isolated after RP-HPLC purification
and lyophilisation.
[0331] MS: m/z 784.4=[M+H].sup.+, (calculated=785.1 g/mol).
[0332] 11 mg of the intermediate were used for deprotection of the
thiol. For deprotection the intermediate was dissolved in 1.2 mL
HFIP/TFA (1/1), 48 .mu.L of TES/water (1/1) was added, and the
solution was agitated for 1.5 h. Volatiles were removed and the
product was purified by RP-HPLC.
[0333] Yield: 7.7 mg (0.011 mmol, double TFA salt).
[0334] MS: m/z 442.2=[M+H].sup.+, (calculated=442.7 g/mol).
Example 5
Synthesis of Linker Pramipexole Conjugate (5b)
Synthesis of Intermediate (5a)
##STR00024##
[0336] Boc-Gly-OH (659 mg, 3.76 mmol), PyBOP (2.35 g, 4.51 mmol)
and N-methyl morpholine (4.14 mL, 37.6 mmol) were dissolved in DMSO
(20 mL). Pramipexole dihydrochloride (2.14 g, 7.52 mmol) were
added, and the mixture was stirred for 1 h. The solution was
diluted with 300 mL 1 M NaOH solution, saturated with NaCl, and
extracted with DCM (8.times.70 mL). The combined organic phases
were dried over MgSO.sub.4, the solvent was evaporated under
reduced pressure, and the residue purified by RP-HPLC. After
lyophilisation 721 mg (1.49 mmol, TFA salt) of the Boc protected
derivative were obtained.
[0337] MS: m/z 369.2=[M+H].sup.+, (calculated=369.5 g/mol).
[0338] For deprotection the intermediate was dissolved in 3 M
methanolic HCl (10 mL), concentrated aqueous HCl (400 .mu.L) were
added, and the mixture was agitated for 4 h. The solvent was
removed under reduced pressure and the residue was dried in
vacuo.
[0339] Yield: 490 mg (1.44 mmol, double HCl salt).
[0340] MS: m/z 269.1=[M+H].sup.+ (calculated=269.4 g/mol).
Synthesis of (5b)
##STR00025##
[0342] 6-(Tritylthio)hexane-1-amine (1.21 g, 3.22 mmol) and
p-nitrophenyl chloroformate (0.78 g, 3.86 mmol) were suspended in
dry THF (15 mL). DIEA (841 .mu.L, 4.83 mmol) was added, and the
resulting solution was stirred at room temperature for 2 h. After
acidification by addition of acetic acid the solvent was evaporated
under reduced pressure, and the residue was purified by RP-HPLC.
1.21 g (2.25 mmol) p-nitrophenyl carbamate were obtained after
lyophilisation.
[0343] The carbamate (801 mg, 1.48 mmol) was dissolved in DMSO (4.4
mL) and added dropwise to a stirred solution of 5a (490 mg, 1.44
mmol) and DIEA (800 .mu.L, 4.60 mmol) in DMSO (7 mL) within 30 min.
The mixture was agitated for 4.5 h at room temperature. The
solution was diluted with 0.5 M NaOH solution (300 mL) and
extracted with DCM (6.times.70 mL). The combined organic phases
were dried over MgSO.sub.4, the solvent was evaporated under
reduced pressure, and the conjugate was purified by RP-HPLC to
obtain 254 mg (0.323 mmol, TFA salt) of the trityl protected
intermediate.
[0344] MS: m/z 670.3=[M+H].sup.+ (calculated=671.0 g/mol).
[0345] For deprotection the intermediate (248 mg, 0.32 mmol) was
incubated in HFIP (6 mL) and TES (240 .mu.L) for 30 min at room
temperature. Volatiles were evaporated, and the residue was
purified by RP-HPLC.
[0346] Yield: 167 mg (0.31 mmol, TFA salt).
[0347] MS: m/z 428.2=[M+H].sup.+, (calculated=428.6 g/mol).
Example 6
Synthesis of (6)
##STR00026##
[0349] For the synthesis of intermediate 6 glutaric acid anhydride
(401 mg, 3.52 mmol), pramipexole dihydrochloride (200 mg, 0.70
mmol), and pyridine (567 .mu.L, 7.04 mmol) were dissolved in dry
DMSO (2 mL). The mixture was stirred for 18 hours. The mixture was
acidified by addition of acidic acid and 6 was purified by
RP-HPLC.
[0350] Yield: 191 mg (0.43 mmol, TFA salt).
[0351] MS: m/z 326.2=[M+H].sup.+, (calculated=326.4 g/mol).
Example 7
Synthesis of OEG-Carrier (7)
##STR00027##
[0353] Maleimide-dPEG.sub.6-NHS-ester (75 mg, 0.125 mmol) was
dissolved in 7/3 acetonitrile/water (3 mL). 0.5 M phosphate buffer
pH 7.0 (300 .mu.L) and glycine amide hydrochloride (41 mg, 0.374
mmol) were added, and the solution was aggitated 30 min at RT. The
mixture was diluted by addition of water (3 mL) and 7 purified by
RP-HPLC.
[0354] Yield: 54 mg (0.096 mmol).
[0355] MS: m/z 561.3=[M+H].sup.+, (calculated=561.6 g/mol).
Example 8
Synthesis of OEG-Carrier-Resin (8)
##STR00028##
[0357] PEG-carrier 8 was synthesized on Sieber amide resin (600 mg,
0.38 mmol) by loading of the resin with Fmoc-Phe-OH,
Fmoc-deprotection, coupling with Fmoc-8-amino-3,6-dioxa-octanoic
acid, Fmoc-deprotection, second coupling with
Fmoc-8-amino-3,6-dioxa-octanoic acid and Fmoc-deprotection as
depicted above and described in "Materials and Methods".
[0358] Correct product was confirmed by cleavage of a small amount
of resin as described in "Materials and Methods" and MS
analysis.
[0359] MS: m/z 906.5=[M+H].sup.+ (calculated=906.5 g/mol).
Example 9
Synthesis of PEG-Pramipexole Conjugates (9a), (9b), (9c), (9d), and
(9e)
##STR00029##
[0361] 7 (4.5 mg, 0.008 mmol) and 1b (2 mg, 0.004 mmol) were
dissolved in 1/1 acetonitrile/water (197 .mu.L). 0.5 M phosphate
buffer pH 7.4 (23 .mu.L) was added and the solution aggitated for
10 min at RT. The mixture was acidified by addition of acetic acid,
diluted with water (200 .mu.L), and 9a was purified by RP-HPLC.
[0362] Yield: 3.2 mg (0.003 mmol, TFA salt).
[0363] MS: m/z 931.4=[M+H].sup.+, (calculated=932.1 g/mol).
[0364] 9b was synthesized as described for 9a except for the use of
2b (1.8 mg, 0.003 mmol) instead of 1b.
[0365] Yield: 3.3 mg (0.003 mmol, TFA salt).
[0366] MS: m/z 987.5=[M+H].sup.+, (calculated=987.5 g/mol).
[0367] 9c was synthesized as described for 9a except for the use of
3b (2 mg, 0.004 mmol) instead of 1b.
[0368] Yield: 4.2 mg (0.004 mmol, TFA salt).
[0369] MS: m/z 1003.5=[M+H].sup.+, (calculated=1004.3 g/mol).
[0370] 9d was synthesized as described for 9a except for the use of
4b (2 mg, 0.003 mmol) instead of 1b.
[0371] Yield: 1.8 mg (0.0015 mmol, double TFA salt).
[0372] MS: m/z 1002.5=[M+H].sup.+, (calculated=1003.3 g/mol).
[0373] 9e was synthesized as described for 9a except for the use of
5b (2 mg, 0.004 mmol) instead of 1b.
[0374] Yield: 3.4 mg (0.003 mmol, TFA salt).
[0375] MS: m/z 988.5=[M+H].sup.+, (MW calculated=989.3 g/mol).
Example 10
Synthesis of OEG-Linker Pramipexole Conjugate (10)
##STR00030##
[0377] For the synthesis of PEG-pramipexole conjugate 10 resin 8
(25 mg, 0.012 mmol) was coupled with 6 (5.4 mg, 0.012 mmol) and the
product was cleaved from the resin as described in "Materials and
Methods". 10 was purified by RP-HPLC.
[0378] Yield: 3.9 mg (0.003 mmol, TFA salt).
[0379] MS: m/z 1213.7=[M+H] (calculated=1213.9 g/mol).
Example 11
Synthesis of Backbone Reagents (12g) and (12h)
##STR00031##
[0381] Backbone reagent 12g was synthesized from Amino 4-arm
PEG5000 12a according to following scheme:
##STR00032##
[0382] For synthesis of compound 12b, 4-Arm-PEG5000 tetraamine 12a
(MW ca. 5200 g/mol, 5.20 g, 1.00 mmol, HCl salt) was dissolved in
20 mL of DMSO (anhydrous). Boc-Lys(Boc)-OH (2.17 g, 6.25 mmol) in 5
mL of DMSO (anhydrous), EDC HCl (1.15 g, 6.00 mmol), HOBt.H.sub.2O
(0.96 g, 6.25 mmol), and collidine (5.20 mL, 40 mmol) were added.
The reaction mixture was stirred for 30 min at RT.
[0383] The reaction mixture was diluted with 1200 mL of
dichloromethane and washed with 600 mL of 0.1 N H.sub.2SO.sub.4
(2.times.), brine (1.times.), 0.1 M NaOH (2.times.), and 1/1 (v/v)
brine/water (4.times.). Aqueous layers were reextracted with 500 mL
of DCM. Organic phases were dried over Na.sub.2SO.sub.4, filtered
and evaporated to give 6.3 g of crude product 12b as colorless oil.
Compound 12b was purified by RP-HPLC.
[0384] Yield 3.85 g (59%) colorless glassy product 12b.
[0385] MS: m/z 1294.4=[M+5H].sup.5+ (calculated=1294.6).
[0386] Compound 12c was obtained by stirring of 3.40 g of compound
12b (0.521 mmol) in 5 mL of methanol and 9 mL of 4 N HCl in dioxane
at RT for 15 min. Volatiles were removed in vacuo. The product was
used in the next step without further purification.
[0387] MS: m/z 1151.9=[M+5H].sup.5+ (calculated=1152.0).
[0388] For synthesis of compound 12d, 3.26 g of compound 12c (0.54
mmol) were dissolved in 15 mL of DMSO (anhydrous). 2.99 g
Boc-Lys(Boc)-OH (8.64 mmol) in 15 mL DMSO (anhydrous), 1.55 g EDC
HCl (8.1 mmol), 1.24 g HOBt.H.sub.2O (8.1 mmol), and 5.62 mL of
collidine (43 mmol) were added. The reaction mixture was stirred
for 30 min at RT.
[0389] Reaction mixture was diluted with 800 mL DCM and washed with
400 mL of 0.1 N H.sub.2SO.sub.4 (2.times.), brine (1.times.), 0.1 M
NaOH (2.times.), and 1/1 (v/v) brine/water (4.times.). Aqueous
layers were reextracted with 800 mL of DCM. Organic phases were
dried with Na.sub.2SO.sub.4, filtered and evaporated to give a
glassy crude product.
[0390] Product was dissolved in DCM and precipitated with cooled
(-18.degree. C.) diethylether. This procedure was repeated twice
and the precipitate was dried in vacuo.
[0391] Yield: 4.01 g (89%) colorless glassy product 12d, which was
used in the next step without further purification.
[0392] MS: m/z 1405.4=[M+6H].sup.6+ (calculated=1405.4).
[0393] Compound 12e was obtained by stirring a solution of compound
12d (3.96 g, 0.47 mmol) in 7 mL of methanol and 20 mL of 4 N HCl in
dioxane at RT for 15 min. Volatiles were removed in vacuo. The
product was used in the next step without further purification.
[0394] MS: m/z 969.6=[M+7H].sup.7+ (calculated=969.7).
[0395] For the synthesis of compound 12f, compound 12e (3.55 g,
0.48 mmol) was dissolved in 20 mL of DMSO (anhydrous).
Boc-Lys(Boc)-OH (5.32 g, 15.4 mmol) in 18.8 mL of DMSO (anhydrous),
EDC HCl (2.76 g, 14.4 mmol), HOBt.H.sub.2O (2.20 g, 14.4 mmol), and
10.0 mL of collidine (76.8 mmol) were added. The reaction mixture
was stirred for 60 min at RT.
[0396] The reaction mixture was diluted with 800 mL of DCM and
washed with 400 mL of 0.1 N H.sub.2SO.sub.4 (2.times.), brine
(1.times.), 0.1 M NaOH (2.times.), and 1/1 (v/v) brine/water
(4.times.). Aqueous layers were reextracted with 800 mL of DCM.
Organic phases were dried over Na.sub.2SO.sub.4, filtered and
evaporated to give crude product 12f as colorless oil.
[0397] Product was dissolved in DCM and precipitated with cooled
(-18.degree. C.) diethylther. This step was repeated twice and the
precipitate was dried in vacuo.
[0398] Yield 4.72 g (82%) colourless glassy product 12f which was
used in the next step without further purification.
[0399] MS: m/z 1505.3=[M+8H].sup.8+ (calculated=1505.4).
[0400] Backbone reagent 12g was obtained by stirring a solution of
compound 12f (MW ca 12035 g/mol, 4.72 g, 0.39 mmol) in 20 mL of
methanol and 40 mL of 4 N HCl in dioxane at RT for 30 min.
Volatiles were removed in vacuo.
[0401] Yield 3.91 g (100%), glassy product backbone reagent
12g.
[0402] MS: m/z 977.2=[M+9H].sup.9+ (calculated=977.4).
Synthesis of Backbone Reagent 12h
##STR00033##
[0404] Backbone reagent 12h was synthesized as described for 12g
except for the use of 4-arm PEG2000 instead of 4-arm PEG5000.
[0405] MS: m/z 719.4=[M+9H].sup.8+ (calculated=719.5).
Example 12
Synthesis of Crosslinker Reagents (13d), (13e), and (13f)
[0406] Crosslinker reagent 13d was prepared from adipic acid mono
benzyl ester (English, Arthur R. et al., Journal of Medicinal
Chemistry, 1990, 33(1), 344-347) and PEG2000 according to the
following scheme:
##STR00034##
[0407] A solution of PEG2000 (13a) (11.0 g, 5.5 mmol) and benzyl
adipate half-ester (4.8 g, 20.6 mmol) in dichloromethane (90.0 mL)
was cooled to 0.degree. C. Dicyclohexylcarbodiimide (4.47 g, 21.7
mmol) was added followed by a catalytic amount of DMAP (5 mg) and
the solution was stirred and allowed to reach room temperature
overnight (12 h). The flask was stored at +4.degree. C. for 5 h.
The solid was filtered and the solvent completely removed by
destillation in vacuo. The residue was dissolved in 1000 mL
1/1(v/v) ether/ethyl acetate and stored at RT for 2 hours while a
small amount of a flaky solid was formed. The solid was removed by
filtration through a pad of Celite.RTM.. The solution was stored in
a tightly closed flask at -30.degree. C. in the freezer for 12 h
until crystallisation was complete. The crystalline product was
filtered through a glass frit and washed with cooled ether
(-30.degree. C.). The filter cake was dried in vacuo. Yield: 11.6 g
(86%) 13b as a colorless solid. The product was used without
further purification in the next step.
[0408] MS: m/z 813.1=[M+3H].sup.3+ (calculated=813.3)
[0409] In a 500 mL glass autoclave PEG2000-bis-adipic
acid-bis-benzyl ester 13b (13.3 g, 5.5 mmol) was dissolved in ethyl
acetate (180 mL) and 10% Palladium on charcoal (0.4 g) was added.
The solution was hydrogenated at 6 bar, 40.degree. C. until
consumption of hydrogen had ceased (5-12 h). Catalyst was removed
by filtration through a pad of Celite.RTM. and the solvent was
evaporated in vacuo. Yield: 12.3 g (quantitative) 13c as yellowish
oil. The product was used without further purification in the next
step.
[0410] MS: m/z 753.1=[M+3H].sup.3+ (calculated=753.2)
[0411] A solution of PEG2000-bis-adipic acid half ester 13c (9.43
g, 4.18 mmol), N-hydroxysuccinimide (1.92 g, 16.7 mmol) and DCC
(3.44 g, 16.7 mmol) in 75 mL of DCM (anhydrous) was stirred over
night at room temperature. The reaction mixture was cooled to
0.degree. C. and precipitate was filtered off. DCM was evaporated
and the residue was recystallized from THF.
[0412] Yield: 8.73 g (85%) crosslinker reagent 13d as colorless
solid.
[0413] MS: m/z 817.8=[M+3H].sup.3+ (calculated=817.9).
Synthesis of 13e
##STR00035##
[0415] 13e was synthesized as described for 13d except for the use
of glutaric acid instead of adipic acid
[0416] MS: m/z 764.4=[M+3H].sup.3+ (calculated=764.5).
Synthesis of 13f
##STR00036##
[0418] 13f was synthesized as described for 13d except for the use
of PEG600 instead of PEG2000
[0419] MS: m/z 997.5=[M+H].sup.+ (calculated=997.8)
Example 13
Preparation of Hydrogel Beads (14a), (14b), and (14c) Containing
Free Amino Groups
[0420] A solution of 300 mg 12g and 900 mg 13d in 10.8 mL DMSO was
added to a solution of 100 mg Arlacel P135 (Croda International
Plc) in 60 mL heptane. The mixture was stirred at 700 rpm with a
custom metal stirrer for 10 min at RT to form a suspension. 1.1 mL
N,N,N',N'-tertramethylene diamine (TMEDA) was added to effect
polymerization. After 2 h, the stirrer speed was reduced to 400 rpm
and the mixture was stirred for additional 16 h. 1.6 mL of acetic
acid were added and then after 10 min 50 mL of water were added.
After 5 min, the stirrer was stopped and the aqueous phase was
drained.
[0421] For bead size fractionation, the water-hydrogel suspension
was wet-sieved on 75, 50, 40, 32 and 20 .mu.m steel sieves. Bead
fractions that were retained on the 32, 40, and 50 .mu.m sieves
were pooled and washed 3 times with water, 10 times with ethanol
and dried for 16 h at 0.1 mbar to give 14a as a white powder.
[0422] 14b was prepared as described for 14a except for the use of
322 mg 12h, 350 mg 13f, 2.9 ml DMSO, 1.6 ml TMEDA, 2.4 ml acetic
acid and a stirring speed of 1000 rpm.
[0423] 14c was prepared as described for 14a except for the use of
300 mg 12g, 810 mg 13e, 6.3 ml DMSO, 1.1 ml TMEDA, 1.6 ml acetic
acid and a stirring speed of 1000 rpm.
Example 14
Preparation of Maleimide Functionalized Hydrogel Beads (15a) and
(15b) and Determination of Maleimide Substitution
[0424] A solution of 600 mg Mal-dPEG.sub.6-NHS (1.0 mmol) in 4.5 mL
2/1 (v/v) acetonitrile/water was added to 200 mg dry hydrogel beads
14a. 500 .mu.L sodium phosphate buffer (pH 7.4, 0.5 M) was added
and the suspension was agitated for 30 min at room temperature.
Beads 15a were washed five times each with 2/1 (v/v)
acetonitrile/water, methanol and 1/1/0.001 (v/v/v/)
acetonitrile/water/TFA.
[0425] For determination of maleimide content, an aliquot of
hydrogel beads 15a was lyophilized and weighed out. Another aliquot
of hydrogel beads 15a was reacted with excess mercaptoethanol (in
50 mM sodium phosphate buffer, 30 min at RT), and mercaptoethanol
consumption was detected by Ellman test (Ellman, G. L. et al.,
Biochem. Pharmacol., 1961, 7, 88-95). Maleimide content was
determined to be 0.27 mmol/g dry hydrogel.
[0426] 15b was prepared as described above except for the use of
14b instead of 14a.
[0427] Loading 15b: 0.9 mmol/g
Example 15
Synthesis of Hydrogel-Linker-Pramipexole Conjugate (16a) and
(16b)
##STR00037##
[0429] Maleimide-derivatized hydrogel microparticles 15a (100
.mu.L. loading 30 .mu.mol/mL, 3 mmol) were reacted with compound 5b
(2.3 mg, 4.3 .mu.mol) in 1/1 acetonitrile/water (420 .mu.L) and 0.5
M phosphate buffer pH 7.4 (52 .mu.L) for 10 min at RT. The hydrogel
was washed 20 times with 1/1 acetonitrile/water. Remaining
maleimides where reacted with 2-mercaptoethanol (34 .mu.L, 0.48
mmol) in 1/1 acetonitrile/water (3 mL) and 0.5 M phosphate buffer
pH 7.4 (0.4 mL) for 10 min at RT. The loaded hydrogel was washed 20
times with 1/1 acetonitrile/water, 20 times with phosphate buffer
pH 7.4 and incubated in the same buffer (1.5 mL) at 37.degree.
C.
[0430] Pramipexole loading 16a: 27 mg/g
[0431] High loaded pramipexole linker hydrogel 16b was prepared as
described above except for the use of 88 mg 5b and 100 mg 14b.
[0432] Pramipexole loading 16b: 152 mg/g
Example 16
Release Kinetics In Vitro
[0433] Release of drug molecule from 9a, 9b, 9c, 9d, 9e, 10, 16a,
and 16b was effected by hydrolysis in buffer at pH 7.4 and
37.degree. C.
[0434] 9a, 9b, 9c, 9d, 9e, and 10, respectively, were dissolved in
buffer (60 mM sodium phosphate, 3 mM EDTA, 0.01% Tween20, pH 7.4),
solution was filtered through a 0.2 .mu.m filter and incubated at
37.degree. C. Samples were taken at time intervals and analyzed by
RP-HPLC at 263 nm and 280 nm and ESI-MS. UV-signals correlating to
linker conjugate molecules were integrated and plotted against
incubation time. Curve-fitting software was applied to estimate the
corresponding half time of release
[0435] 16a and 16b, respectively, were suspended in buffer (60 mM
sodium phosphate, 3 mM EDTA, 0.01% Tween20, pH 7.4) and incubated
at 37.degree. C. At time intervals samples were suspended,
centrifuged and samples (10-50 .mu.L) were taken from the
supernatant solution. Samples were diluted with buffer and analyzed
by measurement of the absorption of released drug at 262 nm.
Calculated amounts of released drug were plotted against incubation
time.
[0436] Curve-fitting software was applied to estimate the
corresponding half time of release.
[0437] FIG. 1 additionally depicts the in vitro release kinetic of
the carrier linked pramipexole prodrug of example 16a. The x-axis
shows the time [unit: days].
TABLE-US-00001 Compound t.sub.1/2 buffer A (pH 7.4) 9a 1.2 d 9b 3.7
d 9c 1.3 d 9d 1.1 d 9e 4.3 d 10 100 d 16a 5.1 d 16b 15 d
ABBREVIATIONS
[0438] AcOH acetic acid [0439] Ado 8-amino-3,6-dioxa-octanoic acid
[0440] Boc t-butyloxycarbonyl [0441] DBU
1,3-diazabicyclo[5.4.0]undecene [0442] DCC dicyclohexylcarbodiimide
[0443] DCM dichloromethane [0444] DIC diisopropyl carbodiimide
[0445] DIEA diisopropylethylamine [0446] DMAP
dimethylamino-pyridine [0447] DMF N,N-dimethylformamide [0448] DMSO
dimethylsulfoxide [0449] EDC
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimid [0450] EDTA
ethylenediaminetetraacetic acid [0451] ESI electrospray ionization
[0452] EtOH ethanol [0453] eq stoichiometric equivalent [0454] Fmoc
9-fluorenylmethoxycarbonyl [0455] HATU
O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0456] HFIP hexafluoroisopropanol [0457] HOBt
N-hydroxybenzotriazole [0458] LCMS mass spectrometry-coupled liquid
chromatography [0459] Mal 3-maleimido propionyl [0460]
Mal-dPEG.sub.6-NHS
N-(3-maleimidopropyl)-21-amino-4,7,10,13,16,19-hexaoxa-heneicosanoic
acid NHS ester [0461] MS mass spectrum/mass spectrometry NHS
N-hydroxy succinimide [0462] OEG Oligo(ethylene glycol) [0463] PEG
poly(ethylene glycol) [0464] PyBOP
benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium
hexafluorophosphate [0465] RP-HPLC reversed-phase high performance
liquid chromatography [0466] RT room temperature [0467] TCP
2-chlorotrityl chloride resin [0468] TES triethylsilane [0469] TFA
trifluoroacetic acid [0470] THF tetrahydrofurane [0471] TMEDA
N,N,N',N', tetramethyl ethylene diamine [0472] Trt trityl [0473]
UPLC ultra performance liquid chromatography [0474] UV ultraviolet
[0475] VIS visual
* * * * *
References